Peptides in Inhibiting Insects

ABSTRACT

The subject invention pertains to the use of peptide fragments of cadherins (including cadherin-like proteins). The subject invention includes a cell (and use thereof) comprising a polynucleotide that expresses the peptide fragment. The subject invention includes methods of feeding the peptides to insects. In preferred embodiments, the peptides are fed to target insects together with one or more insecticidal proteins, preferably (but not limited to) B.t. Cry proteins. When used in this manner, the peptide fragment can not only enhance the apparent toxin activity of the Cry protein against the insect species that was the source of the receptor but also against other insect species. Preferably, the cadherin is a  Bacillus thuringiensis  (B.t.) insecticidal crystal protein (Cry) toxin receptor. Preferably, the peptide fragment is a binding domain of the receptor. In some preferred embodiments, the peptide is the binding domain nearest to the membrane proximal ectodomain. Corresponding domains are identifiable in a variety of B.t. toxin receptors.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 11/040,472, filedJan. 21, 2005, which claims priority to provisional application Ser. No.60/538,715, filed Jan. 22, 2004.

GOVERNMENTAL RIGHTS

This invention was made in part with government support under Grant No.AI 29092 awarded by the National Institutes of Health. The governmentmay have certain rights in this invention.

BACKGROUND OF THE INVENTION

Various receptors on insect cells for Bacillus thuringiensis (B.t.)insecticidal toxin proteins are known in the art. See, e.g., U.S. Pat.Nos. 6,586,197; 6,429,360; 6,137,033; and 5,688,691. However, no knownprior art taught or suggested administering fragments of cadherin-likeproteins, especially fragments of B.t. toxin receptors, to insects.

Bacillus thuringiensis as an Insecticide. Bacillus thuringiensis (B. A)is a facultative anaerobic, Gram-positive, motile, spore-formingbacterium. B.t. is accepted as a source of environment-friendlybiopesticide. Farmers have applied B.t. as an insecticidal spray forcontrol of lepidopteran and coleopteran pests for more than 30 years.The United States Environmental Protection Agency has considered B.t.sprays to be so safe that it has exempted them from the requirement of atolerance (a standard for a maximum permissible residue limit on food).

There are other alternatives for delivery of B.t. toxin to targetinsects. B.t. toxin genes are inserted into microorganisms that areassociated with the target insect habitat so that the transformedorganisms will colonize and continue to produce enough quantities oftoxin to prevent insect damage. Examples of these are the insertion ofspecific genes into bacteria that colonize plant leaf surface and rootsexternally, such as Pseudomonas cepacia, or internally, such asClavibacter xyli. However, the release of living recombinantmicroorganisms causes many concerns and regulatory restrictions.Alternative methods of introducing genes into microorganisms have beendeveloped to minimize potential horizontal gene flow to other bacterialspecies. These include using transposase-negative derivatives of Tn5transposon, or suicide vectors that rely on homologous recombination forintegration to be completed. There has also been a development ofnon-viable recombinant organisms that could increase toxin persistencein the field, such as products based on encapsulated B.t. toxins in P.fluorescens. This approach eliminates concerns associated with testingof living genetically engineered microorganisms.

B.t. proteins may be delivered in transgenic plants. Examples of suchplants, called B.t. plants, protected from insect attack include cottonand corn. The U.S. Environmental Protection Agency has approved thecommercial planting of B.t. cotton and corn since 1996.

The mechanism of action of the B.t. toxins proceeds through severalsteps that include solubilization of ingested crystal, proteolyticactivation of the protoxins, binding of toxin to midgut receptors, andinsertion of the toxin into the apical membrane to form ion channels orpores. Binding of the toxin to brush border membrane vesicles (BBMV) issupposed to be a two-step process involving reversible and irreversiblesteps. Multiple receptors may be involved in the process of toxinbinding and membrane insertion.

Tabashnik et al. (Tabashnik 1992) described the phenomenon of synergyfor B.t. Cry toxins and developed a formula for calculating synergy. Cryproteins are considered synergistic if the combined insecticidal potencyis greater than the sum of the individual components. Cry1Aa and Cry1Acare synergistic in bioassays against gypsy moth larvae (Lee and Dean,1996). Other examples of B.t. synergy are reported for the Cry proteinsof B.t. israelensis and combinations of spores and crystals againstPlutella xylostella, the diamondback moth (Liu et al., 1998). Non-B.t.molecules are also known to synergize toxins. For example,ethylenediamine tetra acetic acid (EDTA) synergizes B.t. against P.xylostella. The synergy described herein is novel both in the nature ofthe synergistic molecule and the effect detected on importantLepidoptera larvae.

B.t. Toxin Receptors. Characterization of receptors from insect midgutand investigation of their interaction with Cry toxins provides anapproach to elucidating toxin mode of action and designing improved Crytoxins for pest control. Most Cry toxin-binding midgut proteinsidentified to date belong to two main protein families: cadherin-likeproteins and aminopeptidases. There is in vitro and in vivo evidencesupporting the involvement of aminopeptidases in Cry1 toxicity againstlepidopteran larvae. Aminopeptidases bind Cry toxins specificallyallowing them to form pores in membranes (Masson et al., 1995; Sangadalaet al., 2001; Sangadala et al., 1994). Recent studies provide evidencethat aminopeptidase can function as receptors when expressed in culturedcells (Adang and Luo 2003) and insects (Gill and Ellar 2002; Rajagopalet al., 2002). Aminopeptidases do not always confer susceptibility toCry toxins when expressed in heterologous systems (Banks et al., 2003;Simpson and Newcomb 2000).

Cadherin-like proteins are a class of Cry1 receptor proteins inlepidopteran larvae. Bombyx mori, the silkmoth, has a 175-kDacadherin-like protein called BtR175 that functions as a receptor forCry1Aa and Cry1Ac toxins on midgut epithelial cells (Hara et al., 2003;Nagamatsu et al., 1999). M. sexta has a 210-kDa cadherin-like protein,called Bt-R₁, that serves as a receptor for Cry1A toxins (Bulla 2002a,b; Vadlamudi et al., 1993; Vadlamudi et al., 1995). Bt-R₁ binds Cry1Aa,Cry1Ab, and Cry1Ac toxins on ligand blots (Francis and Bulla 1997).Purified membranes from COS cells expressing Bt-R₁ bound all three Cry1Atoxins in binding assays and ligand blots (Keeton and Bulla 1997).Furthermore, expression of Bt-R₁ on the surface of COS7 cells led totoxin-induced cell toxicity as monitored by immunofluorescencemicroscopy with fixed cells (Dorsch et al., 2002).

Cadherin-like Bt-R₁ protein has been suggested to induce aconformational change in Cry1Ab that allows the formation of a pre-poretoxin oligomer and increases binding affinity for aminopeptidase (Bravoet al. 2004). In Bombyx mori, the cadherin-like protein BtR175 serves asa Cry1Aa receptor (Nagamatsu et al., 1998). Sf9 cells expressing BtR175swell after exposure to Cry1Aa toxin, presumably due to formation of ionchannels in cell membranes (Nagamatsu et al. 1999). When expressed inmammalian COS7 cells, BtR175 induced susceptibility to Cry1Aa (Tsuda etal., 2003).

Hua et al. (Hua et al. 2004) developed a fluorescence-based assay usingDrosophila S2 cells to analyze the function of Manduca sexta cadherin(Bt-R_(1a)) as a Cry1 toxin receptor. Bt-R_(1a) cDNA that differs fromBt-R₁ by 37 nucleotides and two amino acids and expressed it transientlyin Drosophila melanogaster, Schneider 2 (S2) cells (Hua et al. 2004).Cells expressing Bt-R_(1a) bound Cry1Aa, Cry1Ab, and Cry1Ac toxins onligand blots, and in saturation binding assays. More Cry1Ab was boundrelative to Cry1Aa and Cry1Ac, though each Cry1A toxin bound withhigh-affinity (Kd values from 1.7 nM to 3.3 nM). Using fluorescentmicroscopy and flow cytometry assays, (Hua et al. 2004) showed thatCry1Aa, Cry1Ab and Cry1Ac, but not Cry1Ba, killed S2 cells expressingBt-R_(1a) cadherin. These results demonstrated that M. sexta cadherinBt-R_(1a) functions as a receptor for the Cry1A toxins in vivo andvalidates our cytotoxicity assay for future receptor studies.

Involvement of a cadherin-superfamily gene disruption in resistance toCry1Ac has been described for a laboratory resistant strain of Heliothisvirescens (Gahan et al., 2001). The encoded protein, called HevCaLP, hasthe binding properties expected for a Cry1A receptor (Jurat-Fuentes etal. 2004). Similarly, Pectinophora gossypiella larvae with resistancealleles in genes encoding a cadherin-like protein were resistant toCry1A toxins (Morin et al., 2003).

B.t. toxins bind to specific regions on cadherin-like proteins. Regionsof domain II of Cry1A toxins are involved in binding to Bt-R₁ (Gomez etal., 2002; Gomez et al., 2001). The first toxin binding regionidentified in Bt-R₁ was a stretch of seven amino acid residues locatedin the cadherin repeat seven (CR7) (Gomez et al. 2002; Gomez et al.2001). (Dorsch et al. 2002) identified a second Cry1Ab binding regionwithin cadherin repeat 11 (CR11) in Bt-R₁. Recombinant and syntheticpeptides containing both amino acid sequences inhibited Cry1Ab toxicityin vivo when fed to M. sexta larvae (Dorsch et al. 2002; Gomez et al.2001), demonstrating their involvement in toxin action. Previously, twoBt-R₁ toxin-binding regions in CR 7 (Gomez et al. 2001) and 11 (Dorschet al. 2002) were proposed as functional receptor sites. U.S. Ser. No.60/538,753 entitled “Novel Binding Domain of Cadherin-like ToxinReceptor,” by Adang et al., under Attorney Docket No. UGR-104P,identifies an additional binding site recognized by Cry toxins thatfunctions as a receptor. This additional binding site, which is also afunctional receptor region, is contained in the CR12—Membrane ProximalExtracellular Domain (MPED) of Bt-R1_(a) (Hua et al. 2004). The HevCaLPprotein of H. virescens has a Cry1Ac binding site at a comparableposition (Xie et al. 2004), suggesting a conservation of binding sitesbetween cadherins of different insect species.

There is no known report or suggestion of a B.t. toxin receptor orfragment thereof being fed, or otherwise administered, to an insectpest, with or without a B.t. protein, in order to kill or otherwiseprevent the insect from feeding on a plant. Previous competitive-bindingstudies suggest that there would be no change in toxicity (Gomez et al.2002) or a reduction in toxicity due to competitive binding (Gomez etal. 2001; Dorsch et al. 2002; Gomez et al. 2003; Xie et al. 2004).

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to the use of peptide fragments ofproteins for controlling insects. In preferred embodiments, the sourceprotein is a cadherin (including cadherin-like proteins) and/or aBacillus thuringiensis (B.t.) crystal protein (Cry) toxin receptors.Preferably, the peptide fragment is a binding domain of the receptor. Insome preferred embodiments, the peptide is the binding domain nearest tothe membrane proximal ectodomain. Corresponding domains are identifiablein a variety of B.t. toxin receptors. Thus, one aspect of the inventionpertains to the use of an isolated polynucleotide that encodes a proteincomprising (or consisting of) a fragment of a cadherin-like protein.

In preferred embodiments, the peptides are fed to target insectstogether with one or more insecticidal proteins, preferably (but notlimited to) B.t. Cry proteins. When used in this manner, the peptidefragment can not only enhance the apparent toxin activity of the Cryprotein against the insect species that was the source of the receptorbut also against other insect species.

The subject invention includes a cell (and use thereof) carrying thepolynucleotide and expressing the peptide fragment, including methods offeeding the peptide (preferably with B.t. Cry toxins) to insects.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates Bt-R_(1a) truncated cadherin constructs expressed onthe surface of S2 cells using the vector pIZT-V5-His (Invitrogen) andtransfected into Drosophila S2 cells. Plasmids are designated for thecadherin repeats (CR) encoded. Numbers in parentheses indicated theamino acid residues of the CR start and end positions. CR units 7 and 11(in black) contain Toxin Binding Regions 1 and 2, respectively.

FIG. 2 shows results of toxin binding assays under native conditions(dot blotting and binding saturation assays). Designations are accordingto Bt-R_(1a) constructs in FIG. 1. FIG. 2 shows Cry1Ab binding totruncated and full-length Bt-R_(1a) cadherin expressed in Drosophila S2cells under non-denaturing conditions and competition by CR12-MPEDpeptide. S2 cells (5×10⁵ cells) were dot-blotted on PVDF filters. Afterblocking, the filters were probed with ¹²¹I-Cry1Ab or ¹²⁵I-Cry1Ab plus1000-fold excess (molar ratio) purified CR12-MPED peptide. FIG. 2 shows¹²⁵I-Cry1Ab binding to the expressed Cad12 truncated fragment containingCR12, but not to CR11 alone.

FIG. 3 shows the amino acid sequence of CR12-MPED truncated M. sextacadherin Bt-R_(1a) in pET-30a(+) (Novagen). Bold letters and underlinedesignates Bt-R_(1a) amino acids. The truncated open reading frame fromBt-R_(1a) is designated CR12-MPED. (264 residues total-206 residues fromBt-R_(1a) (78%); 58 residues from pET-30a(+) (22%). MW=28652 Dalton.).

FIGS. 4A and 4B illustrate that CR12-MPED enhanced the potency of B.t.Cry1Ab. FIGS. 4A and 4B show live and dead larvae, and illustrate thereduced size of larvae in all groups fed with combinations of Cry 1 Abplus CR12-MPED.

FIGS. 5A-F shows the toxicity effect as body-weight of surviving Manducasexta, Heliothis virescens, Helicoverpa zea, Spodoptera frugiperda, andPlutella xylostella larvae fed CR12-MPED truncated cadherin peptide withCry1A toxins.

FIGS. 6A-F show photographs of surviving Manduca sexta, Heliothisvirescens, Helicoverpa zea, Spodoptera frugiperda, B.t.-susceptiblePlutella xylostella, and B.t.-resistant Plutella xylostella larvae fed amixture of B.t. Cry1A toxins and CR12-MPED truncated cadherin peptidewith Cry1A toxins.

FIG. 7 shows that CR11-MPED enhances Cry1Ab toxicity to Manduca sexta(tobacco hornworm).

FIG. 8 shows bioassay of Cry1Ac with cadherin fragments on soybeanlooper (Pseudoplusia includens).

FIG. 9 shows bioassay on soybean looper with Cry2Aa and differenttruncations of BtR1_(a) cadherin. Ano-PCAP data are included.

FIG. 10A demonstrates that CR12-MPED peptide was able to enhance theactivity of Cry1Aa protoxin, as well as trypsin-digested truncatedCry1Aa (FIG. 10B) against P. includens.

FIG. 11 illustrates results of the diet overlay bioassay on the soybeanlooper (Pseudoplusia includens) neonate mortality to the mixture ofCR12-MPED and 5 ng/cm² Cry1Ac with different toxin:peptide ratios.

FIG. 12 illustrates results of the diet overlay bioassay on the cabbagelooper (Trichoplusia ni) neonate mortality to the mixture of CR12-MPEDand 8 ng/cm² Cry1Ac with different toxin:peptide ratios.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a nucleotide sequence that encodes the CR12-MPED peptide.

SEQ ID NO:2 is the amino acid sequence of the CR12-MPED peptide. Thispeptide may be referred to as “B.t. Booster” or “BTB.”

SEQ ID NO:3 shows the nucleotide sequence of CR11-MPED truncated form ofM. sexta cadherin Bt-R_(1a). CR11-MPED can be referred to as BtB₂, whichhas 324 amino acid residues from Bacillus thuringiensis-R_(1a) encodingan approximately 35,447 Dalton protein (theoretical pI=4.72).

SEQ ID NO:4 shows the amino acid sequence of CR11-MPED truncated form ofM. sexta cadherin Bt-R_(1a). This peptide is as-produced by E. colistrain BL21/DE3/pRIL cloned with the pET-30a vector.

SEQ ID NO:5 shows the nucleotide sequence of CR1-3 from M. sexta BtR1a.

SEQ ID NO:6 shows the amino acid sequence of CR1-3 from M. sexta BtR1a.This peptide is as-produced by E. coli strain BL21/DE3/pRIL cloned withthe pET-30a vector.

SEQ ID NO:7 (file Anof-PCAPseq.doc) shows the nucleotide sequence of theputative cell adhesion protein of Anopheles gambiae (NCBI LOCUSXM_(—)321513).

SEQ ID NO:8 (file Anof-PCAPseq.doc) shows the amino acid sequence of theputative cell adhesion protein of Anopheles gambiae (NCBI LOCUSXM_(—)321513).

SEQ ID NO:9 shows the nucleotide sequence encoding “PCAP”—the truncationfrom the Anopheles gambiae putative cell-adhesion protein (P CAP).

SEQ ID NO:10 shows the truncated PCAP (putative cell-adhesion protein)region of the Anopheles gambiae protein. This truncated peptide isreferred to herein as PCAP or Ano-PCAP (213 amino acid residues—a24416.56 Dalton protein, theoretical pI 4.96). This peptide isas-produced from the DNA being cloned into pET-30a vector and expressedin E. coli strain BL21/DE3/pRIL.

SEQ ID NO:11 shows the full-length Anopheles gambiae cDNA cadhereinsequence. BLAST search with the sequence matches the DNA and predictedprotein sequence for a partial Anopheles gambiae cDNA NCBI LocusXM_(—)312086).

SEQ ID NO:12 shows the “Ano-Cad”-encoding fragment of SEQ ID NO:11 thatwas cloned into the pET-30A vector and expressed in E. coli strainBL21/DE3.

SEQ ID NO:13 shows the full-length Anopheles gambiae cadherin proteinencoded by SEQ ID NO:11. Residues 1358-1569 of SEQ ID NO:13 correspondto the “Ano-Cad” peptide encoded by SEQ ID NO:12.

SEQ ID NO:14 shows the amino acid sequence of CR12-MPED truncated M.sexta cadherin Bt-R_(1a) in pET-30a(+) (Novagen).

DETAILED DESCRIPTION OF THE INVENTION

The subject invention concerns methods and materials used forcontrolling insects and other pests, particularly plant pests. Morespecifically, the subject invention pertains to the use of peptidefragments of a protein, preferably a cadherin (including cadherin-likeproteins), for controlling insects. Alternatively or in addition, theprotein is preferably a Bacillus thuringiensis (B.t.) crystal protein(Cry) toxin receptor. These peptide fragments are provided, or madeavailable, to target pests for ingestion. This can be accomplished by avariety of means that are known in the art, some of which are discussedin more detail below.

Preferred are fragments of the ectodomains of cadherin proteins (theportion of the protein that is outside of the cell when part of thecadherin protein is embedded in the cellular membrane and part isexposed at the cell surface). Preferably, the cadherins can be Bacillusthuringiensis (B.t.) crystal protein (Cry) toxin receptors. Preferably,the peptide fragment is a binding domain of the receptor. In somepreferred embodiments, the peptide is the binding domain nearest to themembrane proximal ectodomain. Corresponding domains are identifiable ina variety of B.t. toxin receptors.

In preferred embodiments, the peptides are fed to target insectstogether with one or more insecticidal proteins, preferably (but notlimited to) B.t. Cry proteins. When used in this manner, the peptidefragment can not only enhance the apparent toxin activity of the Cryprotein against the insect species that was the source of the receptorbut also against other insect species.

A related aspect of the inventions pertains to the use of an isolatedpolynucleotide that encodes a protein comprising (or consisting of) afragment of a cadherin-like protein. The subject invention includes acell (and use thereof) carrying the polynucleotide and expressing thepeptide fragment, including methods of feeding the peptide (preferablywith B.t. Cry toxins) to insects. The nucleotide sequences can be usedto transform hosts, such as plants, to express the receptor fragments(preferably cadherin fragments) of the subject invention. Transformationof plants with the genetic constructs disclosed herein can beaccomplished using techniques well known to those skilled in the art.Thus, in some embodiments, the subject invention provides nucleotidesequences that encode fragments of receptors, preferably a Bt-R,cadherin-like protein.

The receptor used as the source of this domain(s) can be derived fromvarious pests and insects, such as Manduca sexta, Heliothis virescens,Helicoverpa zea Spodoptera frugiperda and Plutella xylostella larvae.Many sequences of such receptors are publicly available. The subjectpeptide fragments can not only enhance a toxin's activity against theinsect species that was the source of the receptor, but also againstother insect species.

Various pests can be targeted, including but not limited to Manducasexta, Heliothis virescens, Helicoverpa zea Spodoptera frugiperda andPlutella xylostella larvae. Because of the unique and novel approach ofthe subject invention, pests that were typically not susceptible to B.t.Cry proteins can now also be targeted. For example, hemipteransrepresent a major group of insects that have typically not beeneffectively controlled by B.t. δ-endotoxins. Numerous hemipteran pestspecies, most notably Lygus species, cause considerable plant damage andeconomic loss each year. The digestive system of hemipterans (includingaphids) is unusual among the insects in several ways: certain hydrolyticdigestive enzymes are absent such as trypsin; the midgut lacks aperitrophic membrane, and there is no crop. These features reflect theliquid diet and sucking mode of feeding, subject to evolutionaryconstraints. However, because of the subject novel approach, the subjectinvention offers new alternatives for pest control. The subjectinvention can be used to enhance and expand the spectrum (or insectrange) of toxicity of a given insect-toxic protein.

In some preferred embodiments, these peptide fragments can be used toenhance the potency of B.t. toxins for controlling insects. In somepreferred embodiments, the peptide fragments enhance the toxicity ofCry1 toxins, but as shown herein, the subject invention is not limitedto use with such toxins.

Various types of plants and crops can be protected in a variety of waysby practicing the subject invention. Cotton and corn are the main cropsthat can be protected by peptides (and proteins) of the subjectinvention, as well as soybeans and rice. Preferred methods forprotecting these crops include producing transgenic crops that areengineered to produce peptides (and proteins) according to the subjectinvention. Preferred uses for spray-on applications include, but are notlimited to, protecting vegetables and targeting forest pests (protectingplanted trees and the like). Preferred pests for targeting in thismanner include but are not limited to lepidopterans.

Without being bound by a specific theory or theories of mechanism ofaction, one possibility is that these fragments work in conjunction withB.t. toxins and enhance the pesticidal activity of the toxin. When fedto insects with a Cry toxin, the peptide can change the effect of atoxin from a growth-inhibitory effect to an insecticidal effect. Inaddition or alternatively, the fragments can exert at least a partialtoxic effect by a separate mechanism of action. Yet another possibilityis that the fragments also, or alternatively, work indirectly tostabilize the B.t. toxin. Thus, said fragment can work independentlyfrom the Cry toxin (by another mechanism of action) and/or inconjunction with the Cry toxin to enhance the insecticidal potency ofthe Cry toxin. However, the mechanisms) of action are not important forpracticing the subject invention. Based on the subject disclosure, oneskilled in the art can practice various aspects of the subject inventionin a variety of ways.

For example, the fragment of cadherin-like protein may be expressed as afusion protein with a B.t. Cry toxin using techniques well known tothose skilled in the art. As described herein, preferred fusions wouldbe chimeric toxins produced by combining a toxin (including a fragmentof a protoxin, for example) and a fragment of a cadherin-like protein.In addition, mixtures and/or combinations of toxins and cadherin-likeprotein fragments can be used according to the subject invention. Thesemixtures or chimeric proteins have the unexpected and remarkableproperties of enhanced insecticidal potency.

It should similarly be noted that one skilled in the art, having thebenefit of the subject disclosure, will recognize that the subjectpeptides potentially have a variety of functions, uses, and activities.As stated herein, the subject peptides can be administered together witha Cry protein. When used in this manner, peptides of the subjectinvention can effect a faster kill of the targeted insects, and/or theycan enable less Cry protein to be required for killing the insects.Complete lethality, however, is not required. The ultimate preferredgoal is to prevent insects from damaging plants. Thus, prevention offeeding is sufficient. Thus “inhibiting” the insects is all that isrequired. This can be accomplished by making the insects “sick” or byotherwise inhibiting (including killing) them so that damage to theplants being protected is reduced. Peptides of the subject invention canbe used alone or in combination with another toxin to achieve thisinhibitory effect, which can be referred to as “toxin activity.” Thus,the inhibitory function of the subject peptides can be achieved by anymechanism of action, directly or indirectly related to the Cry protein,or completely independent of the Cry protein.

In specific embodiments, the subject invention relates to the use of acadherin repeat 12-MPED peptide of Manduca sexta Bt-R_(1a) cadherin-likeprotein to enhance the potency of B.t. toxins. A region (i.e., fragment)of a cadherin-like protein was identified that synergizes theinsecticidal potency of a B.t. Cry toxin. The receptor fragment bindstoxin with high-affinity, catalyzes toxin-induced cell death whenexpressed on the surface of cultured insects cells, and enhances (i.e.,synergizes) the insecticidal potency of a Cry toxin.

However, in light of the subject disclosure, it will be recognized thatother peptides can be used in like manners. For example, a novelCry1Ab-binding site on Bt-R_(1a) was identified as described in U.S.Ser. No. 60/538,753 entitled “Novel Binding Domain of Cadherin-likeToxin Receptor,” by Adang et al., under Attorney Docket No. UGR-104P,which identifies an additional binding site recognized by Cry toxinsthat functions as a receptor. This additional binding site, which isalso a functional receptor region, is contained in the CR12—MembraneProximal Extracellular Domain (MPED) of Bt-R1_(a) (Hua et al. 2004). TheHevCaLP protein of H. virescens has a Cry1Ac binding site at acomparable position (Xie et al. 2004), suggesting a conservation ofbinding sites between cadherins of different insect species.

Full-length and truncated Bt-R_(1a) fragments were geneticallyengineered and expressed in Drosophila S2 cells to test for Cry1Abbinding and cytotoxicity mediated by receptor fragments. See, e.g.,FIG. 1. In toxin binding assays under denaturing conditions (ligandblotting), 125I-Cry1Ab bound to full length Bt-R_(1a), and to Cad7-12,Cad10-12, and Cad11-12 truncated fragments. Binding assays under nativeconditions (dot blotting and binding saturation assays) revealed¹²⁵I-Cry1Ab binding to the expressed Cad12 truncated fragment containingCR12, but not to CR11 alone (See, e.g., FIG. 2). In saturation bindingassays, ¹²⁵I-Cry1Ab toxin bound with similar high affinity to fulllength Bt-R_(1a), Cad7-12, Cad11-12, and Cad12, although theconcentration of receptors was higher for Cad11-12. Fluorescenceassisted cell sorting (FACS) assays showed that S2 cells expressingBt-R_(1a), Cad7-12, Cad10-12, Cad11-12 or Cad12 were susceptible toCry1Ab. S2 cells expressing Cad7 or Cad11 were not killed by the toxin.Thus, described therein is a novel receptor region on Bt-R_(1a) that islocated on CR12. Binding to CR12 is necessary and sufficient to confersusceptibility to Cry1Ab toxin to insect cells.

The subject invention stemmed in part from the unexpected finding that apeptide comprising CR12 and the Membrane Proximal Ectodomain (MPED)(Dorsch et al. 2002) enhanced the toxicity of B.t. Cry1 toxins when fedas a mixture to insect larvae. This peptide, called CR12-MPED, isillustrated in FIG. 3. The peptide not only functions as aCry-toxin-enhancing agent against M. sexta, the original source ofBt-R_(1a) receptor, but functions as an enhancing agent for multipleCry1 toxins against other pest Lepidoptera including H. virescens, H.zea and S. frugiperda. The use of a fragment of a B.t. receptor in thismanner has not heretofore been described or suggested.

In preferred embodiments, the fragment of the receptor is a bindingdomain of the receptor. Without being bound by a specific theoryregarding mechanism of action, binding of this domain to a B.t. toxincould induce a conformational change in the B.t. toxin, thus making itmore toxic, more able to bind the toxin receptor, etc. In some preferredembodiments, the fragment comprises (or consists of) the CR12-MPEDdomain.

The peptides (such as CR12-MPED) and toxins can be fed or otherwiseadministered to the target (insect) pest in various ways, according tothe subject invention. In one preferred embodiment, a transgenic plantproduces the peptide (such as CR12-MPED) and one or more B.t. toxins. Byconsuming the peptide and B.t. protein produced by such plant (e.g., byeating plant tissues and cells containing the peptide and protein), theinsect will thereby contact the peptide and protein. Together, they willexhibit the enhanced toxic effects in the insect gut.

Another preferred method of the subject invention is to spray thepeptide (such as Cad12-MPED) onto transgenic B.t. plants (such as corn,cotton, soybeans, and the like). The peptide can be in a suitablecarrier, as are known in the art. By spraying the peptide in this manneron plant tissues consumed by target pests, the pest will eat both thepeptide (in the spray) and the B.t. protein (produced by and present inthe plant).

Yet another preferred method is to spray both the peptide and the B.t.Cry protein onto plants and the like. Such methods are well-known in theart (but heretofore lacked the synergizing peptides of the subjectinvention). B.t. toxins, and/or the peptide of the subject invention,can be formulated with and agriculturally acceptable carrier, forexample, that is suitable for spray application to plants and the like.

In one embodiment, the subject invention is drawn to the use of apolynucleotide that encodes a CR12 binding domain from M. sexta. In apreferred embodiment, such polynucleotides comprise (or consist of) anucleotide sequence that encodes the CR12-MPED peptide of SEQ ID NO:2.(The N-terminal “G”—glycine residue—for example, can be removed and theremaining fragment of the exemplified sequence can be used, according tothe subject invention.) One such nucleotide sequence is shown in SEQ IDNO:1.

In another embodiment the subject invention is drawn to the use of acell or cells transfected with a polynucleotide molecule that comprisesa nucleotide sequence encoding a CR12-MPED peptide, for example.Further, the protein is preferably, but not necessarily, anchored to andlocalized at the cell membrane, and is capable of binding a toxin. In amore preferred embodiment, said protein mediates an observable toxicityto said cell or cells, including death upon contacting a toxin.

While CR12-MPED is one example referred to above and elsewhere herein,several other peptides are exemplified herein. Some other such peptidesare discussed below in the Examples. Thus, it should be understood thatthese other peptides, and variants thereof, can be referred to in thesame manners as is CR12-MPED.

As described in the background of the invention, many B.t. toxins havebeen isolated and sequenced. Polynucleotides encoding any known B.t.toxins or those yet to be discovered and active fragments thereof (see,for example, U.S. Pat. No. 5,710,020) can be used in accord with theteachings herein. See Crickmore et al. (1998) for a description of otherB.t. toxins. A list of Cry toxins from the Crickmore et al. website isattached as Appendix A. These include, but are not limited to,polynucleotides encoding Cry1A toxins such as Cry1Aa, Cry1Ab, Cry1Ac,preferably, as well as Cry1B, Cry1C, Cry1F, Cry1E, and Cry3A. Cry2toxins are also preferred for co-administration with peptides of thesubject invention. One can also select toxin(s) from the Crickmore list,for example, based on the type of pests being targeted. For example,rootworms were targeted in an Example as discussed below. Thus,anti-rootworm toxins (such as Cry34135 toxins) can preferably be used insuch applications. The subject peptides can also be used to controlmutant insects that are resistant to one or more B.t. toxins. ModifiedCry toxins (such as those described in U.S. Pat. Nos. 6,825,006;6,423,828; 5,914,318; and 5,942,664) can also be used according to thesubject invention B.t. toxins other than Cry toxins (such as “Vip”toxins as categorized in another section of the Crickmore et al.website) are also contemplated for use. Insecticidal proteins fromorganisms other than B.t., such as Bacillus subtilis, are alsocontemplated for use.

In order to provide an understanding of a number of terms used in thespecification and claims herein, the following definitions are provided.

An “isolated” nucleic acid or polynucleotide (or protein) is in a stateor construct that would not be found in nature. Thus, it signifies theinvolvement of “the hand of man.” A polynucleotide encoding a peptide ofthe subject invention, to the extent that the peptide does not occur inthe state of nature, would be an isolated polynucleotide. Thispolynucleotide in a plant genome would also be “isolated” as it is notoccurring in its natural state. The term therefore covers, for example,(a) a DNA which has the sequence of part of a naturally occurringgenomic DNA molecule but is not flanked by both of the coding ornon-coding sequences that flank that part of the molecule in the genomeof the organism in which it naturally occurs; (b) a nucleic acidincorporated into a vector or into the genomic DNA of a prokaryote oreukaryote in a manner such that the resulting molecule is not identicalto any naturally occurring vector or genomic DNA; (c) a separatemolecule such as a cDNA, a genomic fragment, a fragment produced bypolymerase chain reaction (PCR), or a restriction fragment; and (d) arecombinant nucleotide sequence that is part of a hybrid gene, i.e., agene encoding a fusion protein.

A nucleotide sequence is operably linked when it is placed into afunctional relationship with another nucleotide sequence. For instance,a promoter is operably linked to a coding sequence if the promotereffects its transcription or expression. Generally, operably linkedmeans that the sequences being linked are contiguous and, wherenecessary to join two protein coding regions, contiguous and in readingframe. However, it is well known that certain genetic elements, such asenhancers, may be operably linked even at a distance, i.e., even if notcontiguous.

Polynucleotides of the subject invention include an isolatedpolynucleotide “consisting essentially of” a segment that encodes aCR12-MPED peptide, for example, attached to a label or reporter moleculeand may be used to identify and isolate B.t. toxins and the like. Probescomprising synthetic oligonucleotides or other polynucleotides may bederived from naturally occurring or recombinant single or doublestranded nucleic acids or be chemically synthesized. Polynucleotideprobes may be labeled by any of the methods known in the art, e.g.,random hexamer labeling, nick translation, or the Klenow fill-inreaction.

The polynucleotides may also be produced by chemical synthesis, e.g., bythe phosphoramidite method described by Beaucage and Caruthers (1981)Tetra. Letts., 22:1859-1862 or the triester method according to Matteuciet al. (1981) J. Am. Chem. Soc., 103:3185, and may be performed oncommercial automated oligonucleotide synthesizers. A double-strandedfragment may be obtained from the single stranded product of chemicalsynthesis either by synthesizing the complementary strand and annealingthe strand together under appropriate conditions or by adding thecomplementary strand using DNA polymerase with an appropriate primersequence.

DNA constructs prepared for introduction into a prokaryotic oreukaryotic host will typically comprise a replication system (i.e.,vector) recognized by the host, including the intended DNA fragmentencoding the desired polypeptide, and will preferably also includetranscription and translational initiation regulatory sequences operablylinked to the polypeptide-encoding segment. Expression systems(expression vectors) may include, for example, an origin of replicationor autonomously replicating sequence (ARS) and expression controlsequences, a promoter, an enhancer and necessary processing informationsites, such as ribosome-binding sites, RNA splice sites, polyadenylationsites, transcriptional terminator sequences, and mRNA stabilizingsequences. Signal peptides may also be included where appropriate fromsecreted polypeptides of the same or related species, which allow theprotein to cross and/or lodge in cell membranes or be secreted from thecell.

Expression and cloning vectors will likely contain a selectable marker,that is, a gene encoding a protein necessary for the survival or growthof a host cell transformed with the vector. Although such a marker genemay be carried on another polynucleotide sequence co-introduced into thehost cell, it is most often contained on the cloning vector. Only thosehost cells into which the marker gene has been introduced will surviveand/or grow under selective conditions. Typically selection genes encodeproteins that (a) confer resistance to antibiotics or other toxicsubstances, e.g., ampicillin, neomycin, methotrexate, etc.; (b)complement auxotrophic deficiencies; or (c) supply critical nutrientsnot available from complex media. The choice of the proper selectablemarker will depend on the host cell; appropriate markers for differenthosts are known in the art.

It will be recognized by those skilled in the art that the DNA sequencesmay vary due to the degeneracy of the genetic code and codon usage. AllDNA sequences which code for exemplified and/or suggested peptides (andproteins) are included. For example, the subject CR12 peptides areincluded in this invention, including the DNA of SEQ ID NO:1 (plus anATG preceding the coding region), which encodes SEQ ID NO:2. The subjectinvention also includes polynucleotides having codons that are optimizedfor expression in plants, including any of the specific types of plantsreferred to herein. Various techniques for creating plant-optimizedsequences are know in the art.

Additionally, it will be recognized by those skilled in the art thatallelic variations may occur in the DNA sequences which will notsignificantly change activity of the amino acid sequences of thepeptides which the DNA sequences encode. All such equivalent DNAsequences are included within the scope of this invention and thedefinition of the regulated promoter region. The skilled artisan willunderstand that exemplified sequences (such as the CR12-MPED sequence ofSEQ ID NO:1) can be used to identify and isolate additional,non-exemplified nucleotide sequences which will encode functionalequivalents to the sequences given in, or an amino acid sequence ofgreater than 90% identity thereto and having equivalent biologicalactivity. DNA sequences having at least 90%, or at least 95% identity toa recited DNA sequence and encoding functioning peptides (such asCR12-MPED) are considered equivalent sequences and are included in thesubject invention. Other numeric ranges for variant polynucleotides andamino acid sequences are provided below (e.g., 50-99%). Following theteachings herein and using knowledge and techniques well known in theart, the skilled worker will be able to make a large number of operativeembodiments having equivalent DNA sequences to those listed hereinwithout the expense of undue experimentation.

As used herein percent sequence identity of two nucleic acids isdetermined using the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al.(1990) J. Mol. Biol. 215:402-410. BLAST nucleotide searches areperformed with the NBLAST program, score=100, wordlength 12, to obtainnucleotide sequences with the desired percent sequence identity. Toobtain gapped alignments for comparison purposes, Gapped BLAST is usedas described in Altschul et al. (1997) Nucl. Acids. Res. 25:3389-3402.When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (NBLAST and XBLAST) are used. Seencbi.nih.gov website.

Polynucleotides (and the peptides and proteins they encode) can also bedefined by their hybridization characteristics (their ability tohybridize to a given probe, such as the complement of a DNA sequenceexemplified herein). Various degrees of stringency of hybridization canbe employed. The more stringent the conditions, the greater thecomplementarity that is required for duplex formation. Stringency can becontrolled by temperature, probe concentration, probe length, ionicstrength, time, and the like. Preferably, hybridization is conductedunder moderate to high stringency conditions by techniques well known inthe art, as described, for example, in Keller, G. H., M. M. Manak (1987)DNA Probes, Stockton Press, New York, N.Y., pp. 169-170.

As used herein “moderate to high stringency” conditions forhybridization refers to conditions that achieve the same, or about thesame, degree of specificity of hybridization as the conditions “asdescribed herein.” Examples of moderate to high stringency conditionsare provided herein. Specifically, hybridization of immobilized DNA onSouthern blots with ³²P-labeled gene-specific probes was performed usingstandard methods (Maniatis et al). In general, hybridization andsubsequent washes were carried out under moderate to high stringencyconditions that allowed for detection of target sequences with homologyto sequences exemplified herein. For double-stranded DNA gene probes,hybridization was carried out overnight at 20-25° C. below the meltingtemperature (Tm) of the DNA hybrid in 6×SSPE, 5×Denhardt's solution,0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature is describedby the following formula from Beltz et al. (1983).

Tm=81.5° C.+16.6 Log [Na+]+0.41(%G+C)−0.61(%formamide)600/length ofduplex in base pairs.

Washes are typically carried out as follows:

-   -   (1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS        (low stringency wash).    -   (2) Once at Tm-20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS        (moderate stringency wash).

For oligonucleotide probes, hybridization was carried out overnight at10-20° C. below the melting temperature (Tm) of the hybrid in 6×SSPE,5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm foroligonucleotide probes was determined by the following formula fromSuggs et al. (1981):

Tm(° C.)=2(number T/A base pairs)+4(number G/C base pairs)

Washes were typically carried out as follows:

-   -   (1) Twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS        (low stringency wash).    -   (2) Once at the hybridization temperature for 15 minutes in        1×SSPE, 0.1% SDS (moderate stringency wash)

In general, salt and/or temperature can be altered to change stringency.With a labeled DNA fragment of greater than about 70 or so bases inlength, the following can be used:

Low: 1 or 2×SSPE, room temperature

Low: 1 or 2×SSPE, 42° C.

Moderate: 0.2× or 1×SSPE, 65° C.

High: 0.1×SSPE, 65° C.

Duplex formation and stability depend on substantial complementaritybetween the two strands of a hybrid, and, as noted above, a certaindegree of mismatch can be tolerated. Therefore, polynucleotide sequencesof the subject invention include mutations (both single and multiple),deletions, and insertions in the described sequences, and combinationsthereof, wherein said mutations, insertions, and deletions permitformation of stable hybrids with a target polynucleotide of interest.Mutations, insertions, and deletions can be produced in a givenpolynucleotide sequence using standard methods known in the art. Othermethods may become known in the future.

The mutational, insertional, and deletional variants of thepolynucleotide and amino acid sequences of the invention can be used inthe same manner as the exemplified sequences so long as the variantshave substantial sequence similarity with the original sequence. As usedherein, substantial sequence similarity refers to the extent ofnucleotide similarity that is sufficient to enable the variantpolynucleotide to function in the same capacity as the originalsequence. Preferably, this similarity is greater than 50%; morepreferably, this similarity is greater than 75%; and most preferably,this similarity is greater than 90%. The degree of similarity needed forthe variant to function in its intended capacity will depend upon theintended use of the sequence. It is well within the skill of a persontrained in this art to make mutational, insertional, and deletionalmutations that are designed to improve the function of the sequence orotherwise provide a methodological advantage. The identity and/orsimilarity can also be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, or 99% as compared to a sequence exemplified herein.

The amino acid identity/similarity and/or homology will be highest incritical regions of the protein that account for biological activityand/or are involved in the determination of three-dimensionalconfiguration that ultimately is responsible for the biologicalactivity. In this regard, certain amino acid substitutions areacceptable and can be expected if these substitutions are in regionsthat are not critical to activity or are conservative amino acidsubstitutions which do not affect the three-dimensional configuration ofthe molecule. For example, amino acids may be placed in the followingclasses: non-polar, uncharged polar, basic, and acidic. Conservativesubstitutions whereby an amino acid of one class is replaced withanother amino acid of the same type fall within the scope of the subjectinvention so long as the substitution does not materially alter thebiological activity of the compound. Table 1 provides a listing ofexamples of amino acids belonging to each class.

TABLE 1 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

In some instances, non-conservative substitutions can also be made. Thecritical factor is that these substitutions must not significantlydetract from the biological activity of the toxin.

Practicing some embodiments of the subject invention might necessitatethe use of expression vectors comprising one or more polynucleotidescomprising an exemplified nucleic acid sequences, and capable ofexpressing the subject peptides, in a suitable host cell. In the vectorsof the subject invention, the polynucleotide encoding the peptide isoperably linked to suitable transcriptional and/or translationalregulatory elements to effect expression of the peptide in a suitablehost cell. The regulatory elements may be derived from mammalian,microbial, viral or insect genes and include, for example, promoters,enhancers, transcription and translation initiation sequences,termination sequences, origins of replication, and leader and transportsequences. Suitable regulatory elements are selected for optimalexpression in a desired host cell.

Possible regulatory sequences can include, but are not limited to, anypromoter already shown to be constitutive for expression, such as thoseof viral origin (e.g., IEI promoter from Baculoviruses) or so-called“housekeeping” genes (ubiquitin, actin, tubulin) with theircorresponding termination/poly A+ sequences. In addition, the gene canbe placed under the regulation of inducible promoters and theirtermination sequences so that gene expression is induced by light(rbcS-3A, cab-1), heat (hsp gene promoters) or wounding (mannopine,HGPGs). Other suitable promoters include the metallothionein promoter,dexamethasone promoter, alcohol dehydrogenase promoter, and thebaculovirus promoters, i.e., the early promoter (e.g., IE-1 and et1),the late promoters (e.g., vp39 and p6.9), the very late promoters (e.g.,po1 h and p10) and the hybrid promoter (e.g., vp39/po1 h).

It is clear to one skilled in the art that a promoter may be used eitherin native or truncated form, and may be paired with its own or aheterologous termination/polyA+ sequence. In a preferred embodiment, thesubject vectors are regulated by D. melanogaster HSP70 promoter.

Expression vectors can be constructed by well known molecular biologicalmethods as described for example in Sambrook et al. (1989), or any of amyriad of laboratory manuals on recombinant DNA technology that arewidely available. Expression vectors into which the polynucleotides ofthe present invention can be cloned under the control of a suitablepromoter are also commercially available. Recombinant viral vectors,including retroviral, baculoviral, parvoviral and densoviral vectors canbe used but are not particularly preferred. In host cells containingvectors having an inducible promoter controlling the expression of thenucleic acid encoding CR12-MPED, for example, expression is induced bymethods known in the art and suitable for the selected promoter. Forexample, expression of nucleic acids under the control of themetallothionein promoter is induced by adding cadmium chloride or coppersulfate to the growth media of host cells.

In a specific embodiment, the subject invention includes thepest-control use of a host cell containing a vector comprisingnucleotide sequences encoding CR12-MPED under the control of a promoter.The host cell may be procaryotic or eukaryotic, including bacterial,yeast insect and mammalian cells. Insect and mammalian cells arepreferred. Particularly preferred host cells include insect cell lines,such as, for example, Spodoptera frugiperda (Sf and Sf21) andTrichoplusia ni (Tn cells), Estigma acrae (Ea4 cells), Drosophilamelanogaster (Dm cells), Choristoneura fumiferama (Cf-1 cells), Mamestrabrassicae (MaBr-3 cells), Bombyx mori (MnN-4 cells), Helicoverpa zea(Hzlb3 cells), and Lymantria dispar (Ld652Y cells), among others. Thehost cells may be transformed, transfected or infected with theexpression vectors of the present invention by methods well-known tothose of ordinary skill in the art. Transfection may be accomplished byknown methods, such as liposome-mediated transfection, calcium phosphatemediated transfection, microinjection, and electroporation.

Transgenic cells of the subject invention may be obtained bytransfection with a polynucleotide comprising an exemplified (orsuggested) nucleic acid sequence. Equipped with the teachings herein,the skilled artisan would be able to transfect cells with theexemplified, as well as future isolated peptide-encodingpolynucleotides, to produce cells that make peptides of the subjectinvention. Progeny cells that retain the peptide-encoding polynucleotideare, of course, within the scope of the subject invention, as aretransgenic plants.

The term “transfection” as used herein means an introduction of aforeign DNA or RNA into a cell by mechanical inoculation,electroporation, agroinfection, particle bombardment, microinjection, orby other known methods. The term “transformation” as used herein means astable incorporation of a foreign DNA or RNA into the cell that resultsin a permanent, heritable alteration in the cell. Accordingly, theskilled artisan would understand that transfection of a cell may resultin the transformation of that cell.

In preferred embodiments, expression of the peptide- and/ortoxin-encoding gene results, directly or indirectly, in theintracellular production (and maintenance) of the peptide/protein.Plants can be rendered insect-resistant in this manner. Whentransgenic/recombinant/transformed/transfected host cells (or contentsthereof) are ingested by the pests, the pests will ingest the toxicpeptides/proteins. This is one preferred manner in which to causecontact of the pest with the toxin. The result is control (killing ormaking sick) of the pest. Sucking pests can also be controlled in asimilar manner. Alternatively, suitable microbial hosts, e.g.,Pseudomonas such as P. fluorescens, can be applied where target pestsare present; the microbes can proliferate there, and are ingested by thetarget pests.

Where the toxin gene(s) is introduced via a suitable vector into amicrobial host, and said host is applied to the environment in a livingstate, certain host microbes should be used. Microorganism hosts areselected which are known to occupy the “phytosphere” (phylloplane,phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops ofinterest. These microorganisms are selected so as to be capable ofsuccessfully competing in the particular environment (crop and otherinsect habitats) with the wild-type microorganisms, provide for stablemaintenance and expression of the gene expressing the polypeptidepesticide, and, desirably, provide for improved protection of thepesticide from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane(the surface of the plant leaves) and/or the rhizosphere (the soilsurrounding plant roots) of a wide variety of important crops. Thesemicroorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotohacter, Leuconostoc, and Alcaligenes;Fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Ofparticular interest are such phytosphere bacterial species asPseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonasspheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenesentrophus, and Azotobacter vinlandii; and phytosphere yeast species suchas Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C laurentii, Saccharomyces rosei, S.pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus,Kluyveromyces veronae, and Aureobasidium pollulans. Also of interest arepigmented microorganisms.

One aspect of the subject invention is the transformation/transfectionof plants, plant cells, and other host cells with polynucleotides of thesubject invention that express proteins of the subject invention. Plantstransformed in this manner can be rendered resistant to attack by thetarget pest(s).

A wide variety of methods are available for introducing a gene encodinga pesticidal protein into the target host under conditions that allowfor stable maintenance and expression of the gene. These methods arewell known to those skilled in the art and are described, for example,in U.S. Pat. No. 5,135,867.

For example, a large number of cloning vectors comprising a replicationsystem in E. coli/and a marker that permits selection of the transformedcells are available for preparation for the insertion of foreign genesinto higher plants. The vectors comprise, for example, pBR322, pUCseries, M13 mp series, pACYC184, etc. Accordingly, the sequence encodingthe toxin can be inserted into the vector at a suitable restrictionsite. The resulting plasmid is used for transformation into E. coli. TheE. Coli cells are cultivated in a suitable nutrient medium, thenharvested and lysed. The plasmid is recovered. Sequence analysis,restriction analysis, electrophoresis, and other biochemical-molecularbiological methods are generally carried out as methods of analysis.After each manipulation, the DNA sequence used can be cleaved and joinedto the next DNA sequence. Each plasmid sequence can be cloned in thesame or other plasmids. Depending on the method of inserting desiredgenes into the plant, other DNA sequences may be necessary. If, forexample, the Ti or Ri plasmid is used for the transformation of theplant cell, then at least the right border, but often the right and theleft border of the Ti or Ri plasmid T-DNA, has to be joined as theflanking region of the genes to be inserted. The use of T-DNA for thetransformation of plant cells has been intensively researched anddescribed in EP 120 516; Hoekema (1985) In: The Binary Plant VectorSystem, Offset-durkkerij Kanters B. V., Alblasserdam, Chapter 5; Fraleyet al., Crit. Rev. Plant Sci. 4:1-46; and An et al. (1985) EMBO J.4:277-287.

A large number of techniques are available for inserting DNA into aplant host cell. Those techniques include transformation with T-DNAusing Agrobacterium tumefaciens or Agrobacterium rhizogenes astransformation agent, fusion, injection, biolistics (microparticlebombardment), or electroporation as well as other possible methods. IfAgrobacteria are used for the transformation, the DNA to be inserted hasto be cloned into special plasmids, namely either into an intermediatevector or into a binary vector. The intermediate vectors can beintegrated into the Ti or Ri plasmid by homologous recombination owingto sequences that are homologous to sequences in the T-DNA. The Ti or Riplasmid also comprises the vir region necessary for the transfer of theT-DNA. Intermediate vectors cannot replicate themselves in Agrobacteria.The intermediate vector can be transferred into Agrobacteriumtumefaciens by means of a helper plasmid (conjugation). Binary vectorscan replicate themselves both in E. coli and in Agrobacteria. Theycomprise a selection marker gene and a linker or polylinker which areframed by the right and left T-DNA border regions. They can betransformed directly into Agrobacteria (Holsters et al. [1978] Mol. Gen.Genet. 163:181-187). The Agrobacterium used as host cell is to comprisea plasmid carrying a vir region. The vir region is necessary for thetransfer of the T-DNA into the plant cell. Additional T-DNA may becontained. The bacterium so transformed is used for the transformationof plant cells. Plant explants can advantageously be cultivated withAgrobacterium tumefaciens or Agrobacterium rhizogenes for the transferof the DNA into the plant cell. Whole plants can then be regeneratedfrom the infected plant material (for example, pieces of leaf, segmentsof stalk, roots, but also protoplasts or suspension-cultivated cells) ina suitable medium, which may contain antibiotics or biocides forselection. The plants so obtained can then be tested for the presence ofthe inserted DNA. No special demands are made of the plasmids in thecase of injection and electroporation. It is possible to use ordinaryplasmids, such as, for example, pUC derivatives.

The transformed cells grow inside the plants in the usual manner. Theycan form germ cells and transmit the transformed trait(s) to progenyplants. Such plants can be grown in the normal manner and crossed withplants that have the same transformed hereditary factors or otherhereditary factors. The resulting hybrid individuals have thecorresponding phenotypic properties.

In some preferred embodiments of the invention, genes encoding thebacterial toxin are expressed from transcriptional units inserted intothe plant genome. Preferably, said transcriptional units are recombinantvectors capable of stable integration into the plant genome and enableselection of transformed plant lines expressing mRNA encoding theproteins.

Once the inserted DNA has been integrated in the genome, it isrelatively stable there (and does not come out again). It normallycontains a selection marker that confers on the transformed plant cellsresistance to a biocide or an antibiotic, such as kanamycin, G418,bleomycin, hygromycin, or chloramphenicol, inter alia. The individuallyemployed marker should accordingly permit the selection of transformedcells rather than cells that do not contain the inserted DNA. Thegene(s) of interest are preferably expressed either by constitutive orinducible promoters in the plant cell. Once expressed, the mRNA istranslated into proteins, thereby incorporating amino acids of interestinto protein. The genes encoding a toxin expressed in the plant cellscan be under the control of a constitutive promoter, a tissue-specificpromoter, or an inducible promoter.

Several techniques exist for introducing foreign recombinant vectorsinto plant cells, and for obtaining plants that stably maintain andexpress the introduced gene. Such techniques include the introduction ofgenetic material coated onto microparticles directly into cells (U.S.Pat. Nos. 4,945,050 to Cornell and 5,141,131 to DowElanco, now DowAgroSciences, LLC). In addition, plants may be transformed usingAgrobacterium technology, see U.S. Pat. No. 5,177,010 to University ofToledo; U.S. Pat. No. 5,104,310 to Texas A&M; European PatentApplication 0131624B13; European Patent Applications 120516, 159418B1and 176,112 to Schilperoot; U.S. Pat. Nos. 5,149,645, 5,469,976,5,464,763 and 4,940,838 and 4,693,976 to Schilperoot; European PatentApplications 116718, 290799, 320500 all to Max Planck; European PatentApplications 604662 and 627752, and U.S. Pat. No. 5,591,616, to JapanTobacco; European Patent Applications 0267159 and 0292435, and U.S. Pat.No. 5,231,019, all to Ciba Geigy, now Novartis; U.S. Pat. Nos. 5,463,174and 4,762,785, both to Calgene; and U.S. Pat. Nos. 5,004,863 and5,159,135, both to Agracetus. Other transformation technology includeswhiskers technology. See U.S. Pat. Nos. 5,302,523 and 5,464,765, both toZeneca. Electroporation technology has also been used to transformplants. See WO 87/06614 to Boyce Thompson Institute; U.S. Pat. Nos.5,472,869 and 5,384,253, both to Dekalb; and WO 92/09696 and WO93/21335, both to Plant Genetic Systems. Furthermore, viral vectors canalso be used to produce transgenic plants expressing the protein ofinterest. For example, monocotyledonous plant can be transformed with aviral vector using the methods described in U.S. Pat. No. 5,569,597 toMycogen Plant Science and Ciba-Giegy, now Novartis, as well as U.S. Pat.Nos. 5,589,367 and 5,316,931, both to Biosource.

As mentioned previously, the manner in which the DNA construct isintroduced into the plant host is not critical to this invention. Anymethod that provides for efficient transformation may be employed. Forexample, various methods for plant cell transformation are describedherein and include the use of Ti or Ri-plasmids and the like to performAgrobacterium mediated transformation. In many instances, it will bedesirable to have the construct used for transformation bordered on oneor both sides by T-DNA borders, more specifically the right border. Thisis particularly useful when the construct uses Agrobacterium tumefaciensor Agrobacterium rhizogenes as a mode for transformation, although T-DNAborders may find use with other modes of transformation. WhereAgrobacterium is used for plant cell transformation, a vector may beused which may be introduced into the host for homologous recombinationwith T-DNA or the Ti or Ri plasmid present in the host. Introduction ofthe vector may be performed via electroporation, tri-parental mating andother techniques for transforming gram-negative bacteria, which areknown to those skilled in the art. The manner of vector transformationinto the Agrobacterium host is not critical to this invention. The Ti orRi plasmid containing the T-DNA for recombination may be capable orincapable of causing gall formation, and is not critical to saidinvention so long as the vir genes are present in said host.

In some cases where Agrobacterium is used for transformation, theexpression construct being within the T-DNA borders will be insertedinto a broad spectrum vector such as pRK2 or derivatives thereof asdescribed in Ditta et al., (PNAS USA (1980) 77:7347-7351 and EPO 0 120515, which are incorporated herein by reference. Included within theexpression construct and the T-DNA will be one or more markers asdescribed herein which allow for selection of transformed Agrobacteriumand transformed plant cells. The particular marker employed is notessential to this invention, with the preferred marker depending on thehost and construction used.

For transformation of plant cells using Agrobacterium, explants may becombined and incubated with the transformed Agrobacterium for sufficienttime to allow transformation thereof. After transformation, theAgrobacteria are killed by selection with the appropriate antibiotic andplant cells are cultured with the appropriate selective medium. Oncecalli are formed, shoot formation can be encouraged by employing theappropriate plant hormones according to methods well known in the art ofplant tissue culturing and plant regeneration. However, a callusintermediate stage is not always necessary. After shoot formation, saidplant cells can be transferred to medium which encourages root formationthereby completing plant regeneration. The plants may then be grown toseed and said seed can be used to establish future generations.Regardless of transformation technique, the gene encoding a bacterialtoxin is preferably incorporated into a gene transfer vector adapted toexpress said gene in a plant cell by including in the vector a plantpromoter regulatory element, as well as 3′ non-translatedtranscriptional termination regions such as Nos and the like.

In addition to numerous technologies for transforming plants, the typeof tissue that is contacted with the foreign genes may vary as well.Such tissue would include but would not be limited to embryogenictissue, callus tissue types I, II, and III, hypocotyl, meristem, roottissue, tissues for expression in phloem, and the like. Almost all planttissues may be transformed during dedifferentiation using appropriatetechniques described herein.

A variety of selectable markers can be used, if desired. Preference fora particular marker is at the discretion of the artisan, but any of thefollowing selectable markers may be used along with any other gene notlisted herein that could function as a selectable marker.

In addition to a selectable marker, it may be desirous to use a reportergene. In some instances a reporter gene may be used with or without aselectable marker. Reporter genes are genes that are typically notpresent in the recipient organism or tissue and typically encode forproteins resulting in some phenotypic change or enzymatic property. Anassay for detecting reporter gene expression may then be performed at asuitable time after said gene has been introduced into recipient cells.

The skilled artisan will note that polynucleotides preferred forpracticing the subject invention encode proteins (or peptides) capableof expression in cells, localization to cell membrane, and toxinbinding. Accordingly, fragments of exemplified sequences as well asfunctional mutants may equally be used in practicing the subjectinvention. Such fragments and mutants will be readily obtainablefollowing the teachings herein coupled with the state of the art. Forexample, using specifically exemplified polynucleotides as probes,useful polynucleotides can be obtained under conditions of appropriatestringency. Standard hybridization conditions include hybridization withnonspecific DNA, such as salmon DNA, at 50° C. and washing at 45° C. Toobtain polynucleotides having the lowest detectable homology with theexemplified CR12-MPED (for example), hybridization is conducted underconditions of low standard stringency (30-37° C. and 4-6×SSC). Moreclosely related CR12-MPED like polynucleotides (for example) can beobtained under moderate standard stringency conditions (40-50° C. in1×SSC).

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the various described embodimentsare merely exemplary of the present invention and that many apparentvariations thereof are possible without departing from the spirit orscope thereof. Accordingly, one skilled in the art will readilyrecognize that the present invention is not limited to the specificembodiments described herein.

The description provided in the following examples relates to thepreferred method using the available strategy from the publishedprotocols for constructing DNA vectors and the like. Any molecularcloning and recombinant DNA techniques needed would be carried out bystandard methods (Sambrook et al., 1995).

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Summary of Antagonistic Binding and Toxicity Blocking Assays

Previously, toxin binding regions on Bt-R₁ were shown to act asantagonists to Cry1Ab by blocking toxicity (Dorsch et al. 2002; Gomez etal. 2001, Gomez et al. 2003). The CR12-MPED region encoded by the regionof Bt-R1a including cad 12 and the MPED was tested using similarexperiments. The CR12-MPED region (SEQ ID NO:2, encoded by SEQ ID NO:1)was over-expressed in E. Coli and purified. Peptide was mixed with aLC₅₀ dosage of Cry1Ab and fed to M. sexta larvae. Cry1Ab toxin wasobtained by trypsin activation of the protoxin (Accession NumberAAA22330). CR12-MPED was expected to block toxicity when mixed withCry1Ab toxin and fed to larvae. The bioassay results were quitesurprising. The CR12-MPED peptide did not suppress Cry1Ab toxicity butvery surprisingly increased the mortality of Manduca larvae fed Cry1Ab.Increased concentrations of CR12-MPED mixed with a constant amount ofCry1Ab fed to the larvae killed more larvae. CR12-MPED peptide enhancedCry1Ab toxicity. This initial result was confirmed upon further testing.CR12-MPED increases the potency of an already highly active Cry toxinagainst a susceptible insect.

EXAMPLE 2 M. sexta Bioassay Trial 1

“group1”—9 ng/cm2 Cry1Ab (toxin:peptide ratio 1:0),

“group2”—9 ng/cm2 Cry1Ab plus 9 ng/cm2 CR12-MPED (ratio 1:1),

“group3”—9 ng/cm2 Cry1Ab plus 90 ng/cm2 CR12-MPED (ratio 1:10),

“group4”—9 ng/cm2 Cry1Ab plus 450 ng/cm2 CR12-MPED (ratio 1:50),

“group5”—9 ng/cm2 Cry1Ab plus 900 ng/cm2 CR12-MPED (ratio 1:100),

“group6”—9 ng/cm2 Cry1Ab plus 4500 ng/cm2 CR12-MPED (ratio 1:500)

“group7”—water only as a control.

Each group had 16 replicates.

After 7 days, many larvae in groups 4 and 5 were dead. This wasattributed to the possibility that the Tris-HCl buffer, as the CR12-MPEDpeptide was in 10 mM Tris-HCl (pH 8.0). Alternatively, the CR12-MPEDpeptide could have been enhancing the toxicity of Cry1Ab. To determineif the high toxicity of the Cry1Ab/CR12-MPED mixture was reproducible,the bioassay experiment was repeated with additional controls.

EXAMPLE 3 M. sexta Bioassay Trial 2

A 1:500 ratio group (i.e., group 6 above) was not included because the1:50 and 1:100 ratios gave an enhanced effect. Four additional controlswere included: 10 mM Tris-HCl (pH 8.0), 9 ng/cm² CR12-MPED, 90 ng/cm²CR12-MPED, and 900 ng/cm^(,) CR12-MPED. In two days, almost all of thelarvae in highest concentration of CR12-MPED/Cry1Ab were dead, but thelarvae fed with the toxin-only were not dead. The CR12-MPED/Cry1Abtreatment groups showed the same trend as obtained in the first trial.Table 2 shows the percentage mortality for the treatments in both M.sexta trials. CR12-MPED enhanced the potency of B.t. Cry1Ab in bothtrials. FIGS. 4A and 4B show live and dead larvae from Trial 2. Noticethe reduced size of larvae in the all groups fed with combinations ofCry1Ab plus CR12-MPED.

TABLE 2 Bioassay results for Cry1Ab with CR12-MPED to M. sexta larvae. 9ng1Ab/cm 2 + 9 ng1Ab/cm 2 + 9 ng1Ab/cm 2 + 9 ng1Ab/cm 2 + 9 ng1Ab/cm 2 +900 ng 4500 ng 9 ng/cm² 9 ngCR12- 90 ngCR12- 450 ngCR12- CR12- CR12- 1AbMPED MPED MPED MPED MPED Mortality 31.3% 18.8% 56.3% 62.3% 62.5% 100%trial 1 Mortality 31.3% 45.5%  100%  100% trial 2 10 mM 9 ngCR12- 90ngCR12- 900 ngCR12- Tris MPED MPED MPED Mortality   0% trial 1 Mortality16.7% 0% 0% 0% trial 2

EXAMPLE 4 Additional Bioassays

Preliminary data suggested that CR12-MPED synergizes B.t. Cry1Abtoxicity to H. virescens. This is important because H. virescens is themajor target of B.t. cotton.

The ability of the CR12-MPED peptide to function synergistically withother combinations of toxin and pest insects can now be tested, in lightof the subject disclosure.

EXAMPLE 5 Synergistic Effect of CR12-MPED Peptide on Mortality ofHeliothis virescens, Helicoverpa zea, Spodoptera frugiperda, andPlutella xylostella Larvae Fed CR12-MPED Peptide Plus Cry1A Toxins

Eggs were hatched and reared on artificial diet on which toxin or/andCR12-MPED peptide were or not added. Bacillus thuringiensis toxin(Cry1Aa, 1Ab and 1Ac) were used in LC₅₀ dosage according to the Bacillusthuringiensis Toxin Specificity Database (see website atglfc.forestry.ca/bacillus). The three toxins as used were obtained bytrypsin activation of protoxins (Cry1Aa: DH37 (Accession NumberAAA22353); Cry1Ab: NRD12 (Accession Number AAA22330); and Cry1Ac DH73(Accession AAA22331)). The concentration of each toxin is listed in thefollowing tables. H. virescens and H. zea neonates were transferred towells in a bioassay tray containing the diet with or without toxinor/and CR12-MPED peptide. Seven days later, mortality and larval bodyweight were measured. The mortality and body weight were recorded afterseven days feeding with toxins or/and CR12-MPED peptide. Each group hassixteen larvae per treatment. The concentration of CR12-MPED peptide wasin various mass ratios relative to Cry1A toxin as shown in Tables 3-7and in FIGS. 5A-5E. FIGS. 6A-6F are photographs showing survivingManduca sexta, Heliothis virescens, Helicoverpa zeal Spodopterafrugiperda, B.t.-susceptible Plutella xylostella, and B.t.-resistantPlutella xylostella larvae fed a mixture of B.t. Cry1A toxins andCR12-MPED truncated cadherin peptide.

TABLE 3 Manduca Sexta CR12- CR12- CR12- CR12- MPED(0*) MPED(1*)MPED(10*) MPED(100*) H₂O   0%   0%    0% 0% Cry1Aa 6.25% 6.25% 43.75%100% (5.2 ng/cm²) Cry1Ab 31.3% 45.5%   100% 100% (9 ng/cm²) Cry1Ac 37.5%43.75% 93.75% 100% (5.3 ng/cm²) *Values in parentheses designate massratio of CR12-MPED:Cry protein

TABLE 4 Heliothis virescens CR12- CR12- CR12- CR12- MPED(0*) MPED(1*)MPED(10*) MPED(100*) H₂O 0%   0% 0    0% Cry1Aa 6.25%   37.5% 50% 56.25%(52 ng/cm²) Cry1Ab 0% 12.5 50%   75% (0.16 ng/cm²) Cry1Ac 0% 56.25%  75%  100% (4 ng/cm²) *Values in parentheses designate mass ratio ofCR12-MPED:Cry protein

TABLE 5 Helicoverpa zea CR12- CR12- CR12- CR12- MPED(0*) MPED(1*)MPED(10*) MPED(100*) H₂O 0% 0% Cry1Aa 68.75%    70.83% (2.07 ug/cm²)Cry1Ab 0% 33.33% (1.6 ug/cm²) Cry1Ac 50%   62.5% 100% 100% (0.12 ug/cm²)*Values in parentheses designate mass ratio of CR12-MPED:Cry protein

TABLE 6 Spodoptera frugiperda CR12- CR12- CR12- CR12- MPED(0*) MPED(1*)MPED(10*) MPED(100*) H₂O 6.25% 12.5%   0% Cry1Aa 18.75%    0% 18.75%6.25% (50 ng/cm²) Cry1Ab   0%   25%  37.5%   50% (50 ng/cm²) Cry1Ac6.25% 6.25%   50% 62.5% (50 ng/cm²) *Values in parentheses designatemass ratio of CR12-MPED:Cry protein

TABLE 7 Plutella xylostella (non-resistant) CR12-MPED(100*)CR12-MPED(0*) Pupating Mortality Pupating rate Mortality rate H₂O   0%100% 6.25% 87.5% (2 adults) (1 adult) Cry1Aa (1.4 ng/cm²) 7.14% 7.14%81.25% 0% Cry1Ab (3.9 ng/cm²)   25% 31.25%  93.75% 0% Cry1Ac (0.9ng/cm²) 62.5% 12.5% 86.87% 0% *Values in parentheses designate massratio of CR12-MPED:Cry protein

EXAMPLE 6 Theories Regarding Mechanism(s) of Action

Without being bound by specific theories regarding mechanisms of action,following are possible explanations for the “synergistic” or enhancingeffects that peptides of the subject invention have on the insecticidalactivity of B.t. proteins. The peptide may bind to the protein (such asa Cry protein) causing a change in conformation of the toxin, therebyallowing cleavage by midgut proteins and facilitating subsequent bindingand membrane insertion events. A protein/peptide complex could increasebinding to cadherin molecules. In addition, the peptide could increasetoxin binding to receptor molecules such as aminopeptidase or otherraft-associated proteins sorting in the cell membrane. There is evidenceto further support this hypothesis, as cadherin binding increases theaffinity of Cry1Ab for aminopeptidase from M. Sexta (Bravo et al. 2004).With the help of a peptide of the subject invention, the toxin couldgather or collect on the surface of BBMV and form pores on cell surface.The peptides may function as an adaptor or bridge to connect toxin withcell membrane.

Alternatively or in addition, the peptide may function independentlyfrom the Cry toxin, for example. The peptides could exhibit a completeor partial toxic effect elsewhere, separately, or the peptides couldfunction indirectly to enhance the Cry toxin. For example, the peptidecould somehow contribute to the stability of the Cry toxin in the insectgut.

The exact mechanism(s) of action, however, are relatively unimportant,as one skilled in the art can now make and use a wide range ofembodiments of the subject invention as discussed herein.

EXAMPLE 7 Further Studies

7A. Expression and purification of CR12-MPED peptide in E. coli. Twoprimers were designed with restriction sites of Nco I and Xho Iaccording to BtR_(1a) CR12-MPED sequence. CR12-MPED encoding Bt-R_(1a)(¹³⁶²Ile-Pro¹⁵⁶⁷) was cloned into pET-30a(+) vector (Novagen). Thevector pET-30a(+)/CR12-MPED was transferred into E. coli strainBL21/pRIL. Target protein fused with 6×His-tag at both N- and C-terminiwas over-expressed by 1 mM IPTG induction when the culture OD₆₀₀ reached0.5-0.6. The culture was harvested 4 hours after induction. CR12-MPEDpurification was according to “Protocol7” in The QIAexpressionist (2ndEdition, summer 1992, QIAGEN) with minor modifications. The resultingpeptide was dialyzed against 10 mM Tris-HCl (pH 8.0) and confirmed by15% SDS-PAGE and western blot with anti-BtR₁ serum (1:5000). TheCR12-MPED peptide was used in binding competition assays. The PVDFmembrane dotted with S2 cells expressing truncated cadherin wasincubated with CR12-MPED peptide and ¹²⁵I-Cry1Ab toxin in a 500:1 massratio, respectively. CR12-MPED was also tested with Cry1 toxins ininsect bioassays (described below).

7B. Insect bioassays. The LC₅₀ for Cry1Ab against M. sexta neonatelarvae is 5 to 10 ng/cm² (see website: glfc.cfs.nrcan.gc.ca/bacillusOct. 1, 2003). In a bioassay, we confirmed this LC₅₀ value for Cry1Aband selected 9 ng/cm² Cry1Ab for testing the effect of CR12-MPEDpeptide. Toxin preparations were diluted in deionized water, mixed withvarying concentrations of CR12-MPED and then 50 ml applied to thesurface of insect diet (Southland Products, Lake Village, Ark.). M.sexta eggs were obtained from Carolina Biologicals. Mortality was scoredafter 7 days.

H. virescens and Helicoverpa zea eggs were obtained from Benzon Researchand bioassays conducted as for M. sexta.

7C. Results. We expressed full length and truncated peptides of BtR_(1a)in S2 cells to investigate their involvement in Cry1Ab binding andtoxicity. All truncated cadherin constructs contained the signal leaderpeptide as well as the transmembrane and cytoplasmic domain forexpression on the cell membrane. Truncated cadherin fragmentdesignations included the number of the ectodomain cadherin repeats (CR)they contain and the region included. For example Cad7-12 encodes CR7and the remainder of Bt-R_(1a) to the carboxy terminus. Transfected S2cells expressed full length and truncated cadherin fragments, which wererecognized by sera against Bt-R₁ on immunoblots. As previously reported(Hua et al. 2003), S2 cell-expressed full length Bt-R₁ cadherin had aslightly smaller molecular size than Bt-R₁ from M. sexta BBMV.Conversely, the Cad7 and Cad7-12 truncated Bt-R, fragments expressed inS2 cells had a molecular size slightly greater than predicted.

Ligand blots of proteins from S2 transfected cells were probed with¹²⁵I-labeled Cry1Ab toxin. ¹²⁵I-Cry1Ab toxin bound to truncatedBt-R_(1a) that contained Cad7-12, 10-12, and 11-12. Expressed truncatedproteins that did not contain both CR11 and 12 (i.e. Cad7, Cad 11, Cad12and Cad-MPED) did not bind ¹²¹I-Cry1Ab on blots. These results agreedwith previous ligand blot data of truncated Bt-R₁ fragments (Dorsch etal. 2002), which showed Cry1Ab binding to a region that included bothCR11 and 12. To verify if the space between toxin binding region andcell membrane was important for toxin binding, CR11 was switched withCR12 and cloned into pIZT vector. Both dot-blot and ligand-blot showedCad12/11 lost binding with Cry1Ab toxin.

Ligand blotting, which involves denaturing conditions, has been reportedto yield Cry toxin binding results that are sometimes inconsistent withtoxin binding assays done under native conditions (Daniel et al., 2002;Lee and Dean 1996). To investigate the possibility of alteration byligand blotting of binding epitopes that are functional under nativeconditions, we performed dot blotting. S2 cells expressing truncatedcadherin fragments were dotted on PVDF filters and ¹²⁵I-Cry1Ab toxinbinding tested. In agreement with the ligand blot experiments, proteinscontaining Bt-R, ectodomains CR11 and 12 (full length cadherin, Cad7-12,Cad10-12, Cad11-12) specifically bound ¹²⁵I-Cry1Ab. Peptide expressedfrom Cad12 also bound toxins, which was a surprise since it did not bindtoxin on blots. Cad7 and Cad11 did not bind Cry1Ab. Although Cad12-11contained both CR11 and CR12 domains, it did not bind to labeled Cry1Abtoxin after they were switched each other. These results suggest thatthe arrangement among CR11, CR12, and MPED is important for toxinbinding. Interestingly, the expressed Cad12 peptide, which containedonly ectodomain CR12 and MPED, bound Cry1Ab specifically. This resultwas not observed in ligand blotting and is evidence that nativeconditions are necessary for Cry1Ab binding to ectodomain CR12, and thatectodomain CR12 is sufficient for Cry1Ab toxin binding. MPED may also beimportant in maintaining secondary structure of CR12 (a.k.a. EC12) orpossibly collaborates with CR12 in toxin binding. These results identifyectodomain CR12 as a critical Cry1Ab binding epitope on Bt-R_(1a).Interestingly, when radioactivity of the individual dots was counted,the truncated Cad11-12 peptide containing both ectodomain CR11 and 12bound more Cry1Ab toxin than any other expressed truncated peptide,including full length cadherin (data not shown).

To quantify Cry1Ab binding to expressed Bt-R_(1a) fragments, Cry1Abbinding saturation assays were performed with cell suspensions aspreviously reported (Hua et al. 2004). In agreement with results fromdot blotting, cells expressing full-length cadherin, Cad7-12, Cad11-12and Cad12 bound Cry1Ab. Toxin binding was specific and saturable in allcases, and cells expressing cad11-12 bound more toxin than any othercell sample. Although all Bt-R_(1a) fragments binding Cry1Ab displayedthe same binding affinity (Table 8), the concentration of binding siteswas higher for Cad11-12 and Cad12 than for Cad7-12 or full-lengthcadherin. Furthermore, Cad11-12 had about 3-fold higher concentration ofbinding sites than Cad12. These results indicate that in full lengthBt-R1 conformational limitations may exist that prevent maximal bindingof Cry1Ab, and that both CR11 and CR12 contain Cry1Ab binding epitopes.

TABLE 8 Dissociation constants (K_(com)) and concentration of receptors(B_(max)) calculated from ¹²⁵I-Cry1Ab toxin binding saturation assaysBT-R₁ Cad fragment K_(com) (nM) ± error B_(max) (fmoles/mg protein) ±error  7-12 2.05 ± 0.15 505.65 ± 22.09 Cad full 3.55 ± 1.25 625.36 ±14.76 12 2.87 ± 0.84 1407.09 ± 44.73  11-12 3.52 ± 0.99 3319.54 ± 626.94

It was previously reported that Bit-R_(1a) was a functional Cry1A toxinreceptor and induced cell death when expressed in S2 cells (Hua et al.2004). To investigate the role of ectodomains CR11 and 12 in Cry1Abtoxicity, flow cytometry was used to quantitatively measure thepercentage of cytotoxic response induced by Cry1Ab in S2 cellsexpressing different truncated fragments. Co-expression with GFPprovided a method to monitor transfection efficiency, and propidiumiodide (PI) was used to detect dead cells. Cytotoxicity was quantifiedusing a formula previously reported (Hua et al. 2004) that relates bothtransfection and cytotoxicity to background cell death in control (mocktransfected) cells. Cry1Ab was cytotoxic to cells expressing Cad7-12,Cad10-12, Cad11-12, Cad12 and full length Bt-R_(1a) cadherin. On theother hand, Cry1Ab was not toxic to S2 cells expressing Cad7, Cad11,Cad12-11 and Cad-MPED. There were no significant differences among thetoxicities of Cry1Ab on S2 cells expressing Cad7-12, Cad10-12, Cad11-12,Cad12, and full cadherin. These results (summarized in Table 9) areevidence that ectodomain CR12 is the functional receptor epitope forCry1Ab in Bt-R_(1a).

TABLE 9 Summary ¹²⁵I-Cry1Ab binding Construct Denatured native ToxicityCad/full + + + Cad7 − − − Cad7-12 + + + Cad10-12 + + + Cad11-12 + + +Cad12-11 − − NT Cad12 − + + Cad11 − − − Cad-MPED − − −

To confirm the importance of Cad12-MPED region in toxin binding,Cad12-MPED peptide was used as a competitor in dot-blot assays against¹²⁵I-Cry1Ab. Cad12-MPED was expressed in E. coli and purified usingimmobilized metal affinity chromatography. Cad12-MPED peptide competedCry1Ab toxin binding to fill-length cadherin, and truncated cadherins7-12, 10-12, 11-12 and 12. This result was further evidence thatcadherin CR12 domain is necessary and sufficient for toxin binding. CR12contains the key Cry1Ab binding site on Bt-R_(1a) cadherin.

EXAMPLE 8 Summary of Results of Peptide CR12-MPED Enhancing Toxicity ofVarious Cry1A Proteins Against Various Lepidopterans

BtR_(1a) was cloned into the insect cell expression vector pIZT-V5-His(Invitrogen). A fragment of BtR_(1a) extending from cadherin repeat (CR)12 through the membrane proximal extracellular domain (MPED) was clonedinto pET30a and expressed in Escherichia coli. The 27-kDa expressedpeptide called CR12-MPED was partially purified from inclusion bodies.Surprisingly feeding insect larvae CR12-MPED peptide with Cry1 toxinincreased the toxicity of Cry1A toxins to insect larvae.

The CR12-MPED peptide was tested in combination with Cry1A toxinsagainst lepidopteran larvae representing a range of Cry1A toxinsusceptibilities. The following insects were tested: Manduca sexta(tobacco hornworm), Heliothis virescens (tobacco budworm), Helicoverpazea (cotton bollworm, corn earworm), Spodoptera frugiperda (fallarmyworm), and Pseudoplusia includens (soybean looper). Cry1Aa, Cry1Aband Cry1Ac toxins were tested with CR12-MPED using diet-surfacetreatments, early first instar larvae and a 7 day bioassay period.

EXAMPLE 9 Peptide CR12-MPED Enhances Toxicity of Cry1Ab and Cry1AcProteins Against Manduca sexta

In bioassays against M. sexta, CR12-MPED increased mortality from1.0±1.0% for 2 ng Cry1Ab/cm² treatments to 26.0±5.5% mortality at a1:100 Cry1Ab:CR12-MPED mass ratio. As the toxin concentration wasincreased to 4 ng/cm² CR12-MPED increased mortality from 4.2±1.1% to82.3±6.8% (P<0.01). CR12-MPED was inactive alone in all bioassays.

CR12-MPED also enhanced potency of Cry1Ac against M. sexta larvae. Forexample while 2 ng Cry1Ac/cm² killed 13.5±6.5% of the larvae,Cry1Ac:CR12-MPED ratios of 1:10 and 1:100 mortality increased moralityto 63.5±17.8% (P<0.05) and 93.8±3.1% (P<0.005), respectively.

EXAMPLE 10 Peptide CR12-MPED Enhances Toxicity of Cry1Ac AgainstHeliothis virescens

CR12-MPED enhances Cry1Ac toxicity to H. virescens (tobacco budworm).Neonate larvae were fed Cry1Ac with or without CR12-MPED. At a Cry1Acconcentration of 3 ng/cm² diet mortality was 8.4±2.1% (toxin only); withinclusion of 300 ng/cm² of CR12-MPED mortality was increased to83.4±6.3% (P<0.01). At a Cry1Ac concentration of 6 ng/cm², CR12-MPEDpeptide greatly enhanced Cry1Ac toxicity to H. virescens larvae from46.7±9.9% (toxin only) to 88.5±5.5% (with either 1:10 or 1:100 CR12-MPEDratio) (P<0.05).

EXAMPLE 11 Peptide CR12-MPED Enhances Toxicity of Cry1Ac ProteinsAgainst Helicoverpa zea

H. zea (cotton bollworm, corn earworm, tomato fruitworm) has a wide hostrange, attacking many vegetables, fruits, and cotton. B.t. transgenecrops are not as effective at controlling H. zea as they are otherpests. This is because Cry1A toxins are less effective against H. zeathan other target pests. H. zea is not as sensitive to Cry1Ac as H.virescens or M. sexta. Therefore, this pest was selected to determine ifCR12-MPED peptide could enhance B.t. activity.

In this experiment, 50 ng/cm², 60 ng/cm² and 120 ng/cm² of Cry1Ac wereused to test CR12-MPED. Cry1Ac toxin did not kill the larvaeefficiently. At 50 ng or 60 ng/cm² dosages, only 5.2±2.8% of the larvaewere killed, compared to 0% (for toxin only). However, the addition of a1:10 ratio of CR12-MPED increased morality to 56.3±8.3% (P<0.05) and42.7±3.8% (P<0.01). If the toxin dosage was increased to 120 ng/cm², itkilled 24.0±2.8% of H. zea larvae while an equal proportion of addedCR12-MPED increased morality to 47.9±5.5% (P<0.05). A ten-fold amountpeptide increased the mortality to 85.4±2.8 (P<0.001).

EXAMPLE 12 Peptide CR12-MPED Enhances Toxicity of Cry1Ab, Cry1Ac, andCry1C Proteins Against Spodoptera frugiperda

Spodoptera frugiperda is not susceptible to Cry1A toxins (LC₅₀>2000ng/cm² dosage; see, e.g., “glfc.forestry.ca/bacillus” website. However,when Cry1Ab or Cry1Ac were combined with CR12-MPED at 1:1 and 1:10ratios (toxin:CR12-MPED) mortality was increased, and larvae fedcombinations of CR12-MPED and Cry1Ab or Cry1Ac were severely stunted ingrowth S. frugiperda is more susceptible to Cry1Ca (LC₅₀ 1144 (813-3227)ng/cm²) compared to Cry1A toxins. Cry1Ca protoxin (GENBANK AccessionNumber CAA30396), tested at 150 and 300 ng/cm², killed 6% and 19% oflarvae. Addition of CR12-MPED increased morality to 31% and 41%. This isan important observation because there is no published report of Cry1Cainteraction with M. sexta cadherin.

EXAMPLE 13 Summary of CR12-MPED Binding Studies and ConclusionsRegarding Toxin Enhancement

Overall, CR12-MPED enhances or potentiates the toxicity of Cry1A andCry1C toxins against susceptible and tolerant insects. In vitro,CR12-MPED binds to toxin forming large-sized protein clusters. Theseprotein clusters still bind specifically to midgut brush border.

EXAMPLE 14 Peptide CR11-MPED Enhances Toxicity of Cry1Ab Against Manducasexta

The CR11-MPED (SEQ ID NO:4; SEQ ID NO:3 is the DNA) region of BtR_(1a)were cloned into pET30 and expressed in E. coli. The CR11-MPED regionconsists of CR11 in front of CR12-MPED. Peptide was solubilised frominclusion bodies and tested in bioassays with purified Cry1Ab toxin.Peptide CR11-MPED peptide fed with Cry1Ab toxin to M. sexta larvae wasmore toxic to larvae than toxin alone (FIG. 7). The enhancement effectwas dose dependent; increasing with higher ratios of CR11-MPED:Cry1Ab.

EXAMPLE 15 Peptide CR11-MPED Enhances Toxicity of Cry1Ac Against SoybeanLoopers

As discussed in more detail in Example 16, the CR11-MPED peptide alsoincreased Cry1Ac toxicity to soybean loopers (FIG. 8).

EXAMPLE 16 Comparison of the Ability of CR1-3, CR11-MPED, and CR12-MPEDPeptides to Enhance Cry1Ac Against Soybean Loopers

The peptide CR1-3 (SEQ ID NO:6; SEQ ID NO:5 is the DNA) was constructedas a negative control with the expectation that it would not enhancetoxicity based on the lack of a Cry1Ab binding site on the peptide.However, CR1-3 was surprisingly found to be equal to CR12-MPED in thecapacity to increase Cry1Ac toxicity to soybean looper (FIG. 8).

The CR11-MPED and CR1-3 regions of BtR_(1a) were cloned into pET30 andexpressed in E. coli. All peptides were expressed and purified usingstandard methodology. Purified peptides were run on SDS page usingstandard methodology. Concentrations of the indicated samples are asfollows: CR11-MPED (0.279 mg/ml); CR12-MPED (2.106 mg/ml); CR1-3 (1.809mg/ml); Ano-Cad (0.50 mg/ml), Ano-PCAP (0.78 mg/ml) and SlyD (0.046mg/ml). 12.5 ng/cm² of Cry1Ac was bioassayed with or without differentpeptides in 1:1, 1:10 and 1:100 ratio. Soybean looper neonates were setin bioassay trays, with each group having 62 larvae. The Ano-Cad,Ano-PCAP, and SlyD peptides are discussed in the following Example.

EXAMPLE 17 Preparation of Peptides Ano-Cad and Ano-PCAP from MosquitoCadherin Proteins, and Preparation of Peptide SlyD

Mosquitoes are dipterans, as opposed to lepidopterans as tested above.Because selected mosquito cadherin proteins have low amino acidsimilarity to BtR₁, peptide fragments from mosquito cadherins wereexpected to be less likely to enhance a toxin's effect in lepidopteranlarvae, thereby serving as a negative cadherin control. Additionally,mosquito cadherin fragments could be tested for toxin-enhancingproperties in mosquitoes.

Two full-length cDNAs encoding cadherin-type proteins were cloned andsequenced from the mosquito Anopheles gambiae. The cloned cDNAnucleotide sequences correspond to sequences deposited by the Anophelesgambiae genome sequencing project at A. gambiae loci. Fragments similarin size and location to the CR12-MPED region of BtR_(1a) were clonedinto pET30 vector and expressed in E. coli. The cDNA fragments,designated, Ano-Cad and Ano-PCAP, cloned in pET are identical to the DNAsequences of A. gambiae loci XM_(—)312086 and XM_(—)321513,respectively.

SEQ ID NO:7 shows the nucleotide sequence of the putative cell adhesionprotein of Anopheles gambiae NCBI LOCUS XM_(—)321513). SEQ ID NO:8 showsthe corresponding amino acid sequence. SEQ ID NO:9 shows the nucleotidesequence encoding the truncated putative cell-adhesion protein region ofthe Anopheles gambiae protein. This truncated peptide is referred toherein as PCAP putative cell-adhesion protein) or Ano-PCAP, which has213 amino acid residues, and is approximately 24,417 Daltons(theoretical pI=4.96). SEQ ID NO:10 shows the truncated PCAP region ofthe Anopheles gambiae protein. This sequence is for the peptideexpressed in E, coli strain BL21/DE3/pRIL having the DNA cloned into thepET-300a vector.

SEQ ID NO:11 shows the full-length Anopheles gambiae cDNA cadhereinsequence. BLAST search with the sequence matches the DNA and predictedprotein sequence for a partial Anopheles gambiae cDNA (NCBI LocusXM_(—)312086). SEQ ID NO:12 shows the “Ano-Cad”-encoding fragment of SEQID NO:11 that was cloned into the pET-30 vector and expressed in E. colistrain BL21/DE3. SEQ ID NO:13 shows the full-length Anopheles gambiaecadherin protein encoded by SEQ ID NO:11. Residues 1358-1569 of SEQ IDNO:13 correspond to the “Ano-Cad” peptide encoded by SEQ ID NO:12.

SlyD is 21-a kDa histidine-rich E. coli protein that frequentlyco-elutes with other proteins from immobilized metal affinity column(IMAC). Because a similar-sized protein was detected in some eluates,SlyD from E. coli was prepared for testing as well.

EXAMPLE 18 Comparison of the Ability of Ano-PCAP, Ano-Cad, and SlyDPeptides to Enhance Cry1Ac Against Soybean Loopers

As shown in FIG. 8, Ano-PCAP (SEQ ID NO:10) induced some increase intoxicity, whereas the Ano-Cad peptide (residues 1358-1569 of SEQ IDNO:13) did not. SlyD did not have an enhancing effect.

EXAMPLE 19 Comparison of the Ability of Ano-PCAP, CR12-MPED, and CR1-3Peptides to Enhance Cry2Aa Against Soybean Loopers

Cry2Aa protoxin (non-truncated) (GENBANK Accession Number M31738) wasfed to soybean looper larvae (neonates) with CR12-MPED, CR1-3, orAno-PCAP (SEQ ID NO:10). Both CR12-MPED and Ano-PCAP increased Cry2Aatoxicity to the larvae. See FIG. 9. Ratios of toxin:sample are indicatedon the graph. CR1-3 and CR12-MPED were Ni-NTA column elution containing0.25 M imidazole, while Ano-PCAP was purified by ion exchangechromatography (Q sepharose). Cry2Aa protoxin was expressed in E. coliand purified by ion exchange chromatography (Q sepharose). Singleasterisks (*) denotes 0.05<P<0.1 while double asterisks (**) denotesP<0.05 in Chi square statistical calculation comparing toxin onlytreatment with toxin and sample treatment. CR1-3 did not enhance Cry2Aatoxicity. Both CR12-MPED and Ano-PCAP were able to significantly enhanceCry2Aa toxicity.

EXAMPLE 20 Peptides CR11-MPED, CR12-MPED, CR1-3, and Ano-Cad haveStand-Alone Activity Against Rootworms, Cry1Aa Surprisingly has ActivityAgainst Rootworms Coleopterans

A Cry1Aa protein (GENBANK Accession Number AAA22353), which is a toxinproduced in Bacillus thuringiensis (B.t.), was tested as protoxin andtrypsin-treated forms to determine its level of anti-rootworm(Diabrotica spp.; coleopterans) activity, if any. The expectation wasthat this toxin would not be active against this coleopteran, as Cry1toxins (including Cry1Aa) are known to be “lep active” toxins (toxinswith proven activity against caterpillars or lepidopterans). See e.g.Hofte et al. (1989). This protein was surprisingly found to haveactivity against rootworms. Thus, methods of using Cry1Aa forcontrolling rootworms are an aspect of the subject invention.

Surprisingly, in the course of this experimentation, it was also foundthat the CR11-MPED and CR12-MPED peptide have stand-alone activityagainst rootworms. Other cadherin-like peptides eg. CR1-3 and Ano-Cadwere also tested and found to have significant toxicity againstrootworms, albeit at lower toxicity(CR11-MPED≈CCR12-MPED>CR1-3≈Ano-Cad). As the testing and data set forthherein are not exhaustive, the subject invention thus includes the useof peptides of the subject invention, alone, for controlling insects.This methodology is yet another aspect of the subject invention. Inpreferred embodiments of this aspect of the subject invention,“stand-alone” peptides are used to control coleopterans (which includegrubs and beetles).

20.A—Preparation of Cry1Aa, CR11-MPED, CR12-MPED, CR1-3, and Ano-Cad andRootworm Bioassays.

The Cry1Aa construct (cry1Aa gene in pKK223-3 vector) was obtained fromDonald H. Dean (The Ohio State University). Toxin was expressed in E.coli and purified by HPLC. The toxin was concentrated and dialyzedagainst distilled water.

All peptides were over-expressed in E. coli as an inclusion body.Inclusion bodies were extracted from the bacteria and solubilized in 10mM NaOH. Insoluble materials were removed by centrifugation. Thesupernatant was applied to Q-sepharose column (30 mM Na₂CO₃ pH 10.0),and the flow-through fractions containing CR11-MPED were collectedbecause CR11-MPED form large aggregates that fail to bind to the anionexchange column. The fractions were pooled and centrifuged again. Thesupernatant was concentrated by filtration (Amicon) and dialyzed againstdistilled water. Rootworm eggs were purchased from Lee French (FrenchAgricultural Research Inc., Minnesota). Southern Corn Rootworm diet waspurchased from Bio-Serv.

Bioassays were performed on neonate larvae of Southern Corn Rootworm(Diabrotica undecipunctata). The toxin/peptides was diluted in distilledwater and applied on the artificial diet in plastic bioassay trays andair dried. Six larvae were put in each well, and 24 larvae were testedat each toxin dose in Trial 1. In Trial 2, four larvae were put in eachwell, and 16 larvae were tested at each toxin dose. The bioassays weredone at room temperature (23° C.). Mortality was recorded on Day 11 inTrial 1 and Day 10 in Trial 2.

20.8—Toxicity of Cry1Aa CR11-MPED, CR12-MPED, CR1-3, and Ano-Cad on D.undecipunctata.

In Trial 1, 29% mortality was observed at a concentration of 200 μg/cm²Cry1Aa protoxin and 50% mortality was observed at a concentration of 250μg/cm². Higher mortality was recorded for trypsin-treated Cry1Aa. 72%mortality was observed at a concentration of 100 μg/cm² trypsin-treatedCry1Aa and 67% mortality was observed at a concentration of 150 μg/cm².In Trial 2, 100% mortality was achieved with Cry1Aa protoxin at aconcentration of 300 μg/cm². Background mortality was between 6 and 8%in both trials. These results (summarized in Table 10) demonstrated thatCry1Aa has insecticidal activity against rootworms.

TABLE 10 Bioassay results for Cry1Aa protoxin and trypsin-treated toxinto D. undecipunctata larvae 200 μg/cm² 250 μg/cm² Cry1Aa protoxin Cry1Aaprotoxin Mortality Trial 1 29% 50% 100 μg/cm² 150 μg/cm² trypsin-treatedrypsin-treated Cry1Aa t Cry1Aa Distilled H₂O Mortality Trial 1 72%  67%8% 200 μg/cm² 300 μg/cm² Cry1Aa protoxin Cry1Aa protoxin Distilled H₂OMortality Trial 2  0% 100% 6%

The toxicity of the peptides (CR11-MPED, CR12-MPED, CR1-3, and Ano-Cad)towards the rootworm larvae was unexpected because initial tests onsoybean looper larvae showed no toxic activity when the peptides wereapplied alone. Also, a concentration of 100 μg/cm² CR12-MPED did notcause any mortality or growth inhibition to H. zea neonates.

In Trial 1, 25 μg/cm² CR11-MPED killed 8%, 50 μg/cm² CR11-MPED killed8%, 100 μg/cm² CR11-MPED killed 12%, 150 μg/cm² CR11-MPED killed 79%,200 μg/cm² CR11-MPED killed 83%, and 250 μg/cm² CR11-MPED killed 92% ofthe larvae. In the same trial, 25 μg/cm² CR12-MPED killed 8%, 500 μg/cm²CR12-MPED killed 21%, 100 μg/cm² CR12-MPED killed 21%, 150 μg/cm²CR12-MPED killed 96%, 200 μg/cm² CR12-MPED killed 92%, and 250 μg/cm²CR112-MPED killed 83% of the larvae.

In Trial 2, 125 μg/cm² CR11-MPED killed 87%, 150 μg/cm² CR11-MPED killed75%, 200 kg/cm² CR11-MPED killed 88% of the larvae. In the same trial,125 μg/cm² CR1-3 killed 6%, 150 μg/cm² CR1-3 killed 63%, 200 μg/cm²CR1-3 killed 44% of the larvae, while 125 μg/cm² Ano-Cad killed 31%, 150μg/cm² Ano-Cad killed 38%, 200 μg/cm² Ano-Cad killed 50% of the larvae.Background mortality was between 6 and 8% in both trials. These results(summarized in Table 11) demonstrated the stand-alone insecticidalactivity of these peptides against rootworms.

TABLE 11 Bioassay results for CR11-MPED, CR12-MPED, CR1-3, and Ano-Cadto D. undecipunctata larvae 25 μg/cm² 50 μg/cm² 100 μg/cm² 150 μg/cm²200 μg/cm² 250 μg/cm² CR11- CR11- CR11- CR11- CR11- CR11- MPED MPED MPEDMPED MPED MPED Mortality Trial 1 8% 8% 12% 79% 83% 92% 25 μg/cm² 50μg/cm² 100 μg/cm² 150 μg/cm² 200 μg/cm² 250 μg/cm² CR12- CR12- CR12-CR12- CR12- CR12- Distilled MPED MPED MPED MPED MPED MPED H₂O MortalityTrial 1 8% 21% 21% 96% 92% 83% 8% 125 μg/cm² 150 μg/cm² 200 μg/cm² CR11-CR11- CR11- MPED MPED MPED Mortality Trial 2 87% 75% 88% 125 μg/cm² 150μg/cm² 200 μg/cm² CR1-3 CR1-3 CR1-3 Mortality Trial 2 6% 63% 44% 125μg/cm² 150 μg/cm² 200 μg/cm² Distilled Ano-Cad Ano-Cad Ano-Cad H₂OMortality Trial 2 31% 38% 50% 6%

EXAMPLE 21 Mortality of Soybean Looper (Pseudoplusia includens) toMixtures of CR12-MPED and Cry1Aa Protoxin or Trypsin-Digested Cry1Aa

The Cry1Aa construct (cry1Aa gene in pKK223-3 vector) was obtained fromDonald H. Dean (The Ohio State University). Toxin was expressed in E.coli and purified by HPLC. Table 12 shows the diet overlay bioassay onthe soybean looper (Pseudoplusia includens) neonate mortality to Cry1Aain the forms of either protoxin or trypsin-digested truncated toxin, andwith mixtures of CR12-MPED at 1:100 (w/w) ratios. CR12-MPED enhancesCry1Aa toxicity to P. includens. At a Cry1Aa protoxin concentration of 2ng/cm² diet mortality was 23% (toxin only); with inclusion of 200 ng/cm²of CR12-MPED mortality was increased to 50%. At a Cry1Aa protoxinconcentration of 5 ng/cm², 500 ng/cm² CR12-MPED peptide greatly enhancedCry1Aa protoxin toxicity to P. includens larvae from 0% (toxin only) to88% (P<0.001).

Similar results were obtained using trypsin-digested truncated Cry1Aa.At a Cry1Aa trypsin-digested toxin concentration of 2 ng/cm² dietmortality was 6% (toxin only); with inclusion of 200 ng/cm² of CR12-MPEDmortality was increased to 94% (P<0.001). At a Cry1Aa trypsin-digestedtoxin concentration of 5 ng/cm², 500 ng/cm² CR12-MPED peptide enhancedCry1Aa protoxin toxicity to P. includens larvae from 38% (toxin only) to100% (P<0.001). Background mortality was at 0%. These resultsdemonstrated that CR12-MPED peptide was able to enhance the activity ofCry1Aa protoxin (FIG. 10A) as well as trypsin-digested truncated Cry1 Aa(FIG. 10B) against P. includens.

TABLE 12 Bioassay results for Cry1Aa alone and 1:100 ratio (w/w)mixtures of Cry1Aa:CR12-MPED to Pseudoplusia includens larvae 2 ng/cm²Cry1Aa 5 ng/cm² Cry1Aa 10 ng/cm² protoxin protoxin Cry1Aa protoxinMortality 23%  0% 100% 2 ng/cm² Cry1Aa 5 ng/cm² Cry1Aa 10 ng/cm² Cry1Aaprotoxin:200 protoxin:500 protoxin:1000 ng/cm² ng/cm² ng/cm² CR12-MPEDCR12-MPED CR12-MPED Mortality 50% 88% 100% 2 ng/cm² trypsin- 5 ng/cm²trypsin- 10 ng/cm² trypsin- digested Cry1Aa digested Cry1Aa digestedCry1Aa Mortality  6% 38%  75% 2 ng/cm² 5 ng/cm² 10 ng/cm² trypsin-trypsin- trypsin- digested digested digested Cry1Aa:200 Cry1Aa:500Cry1Aa:1000 ng/cm² ng/cm² ng/cm² Distilled CR12-MPED CR12-MPED CR12-MPEDH₂O Mortality 94% 100% 100% 0%

EXAMPLE 22 Mortality of Soybean Looper (Pseudoplusia includens) toVariable Mixtures of CR12-MPED and Cry1Ac

FIG. 11 shows the diet overlay bioassay on the soybean looper(Pseudoplusia includens) neonate mortality to the mixture of CR12-MPEDand 2.5 ng/cm² Cry1Ac (w:w) with different toxin:peptide ratios. Themortality was (5.2±1.1) % when only 2.5 ng/cm² Cry1Ac was applied. Whensame amount of Cry1Ac mixed with CR12-MPED as 1:1 ratio, the neonatemortality was significantly enhanced to (39.9±7.3) % (P<0.05). Themortality was enhanced by approximately 50% when the toxin:peptide ratioreached 1:2 (55.2±8.5) %.

EXAMPLE 23 Mortality of Cabbage Looper (Trichoplusia ni) to VariableMixtures of CR12-MPED and Cry1Ac

FIG. 12 shows the diet overlay bioassay on the cabbage looper(Trichoplusia ni) neonate mortality to the mixture of CR12-MPED and 4ng/cm²Cry1Ac (w:w) with different toxin:peptide ratios. The mortalitywas (10.4±7.5) % when only 4 ng/cm² Cry1Ac was applied. When same amountof Cry1Ac mixed with CR12-MPED as 1:2 ratio, the neonate mortality wassignificantly enhanced to (47.9±13.5) % (P<0.05). The mortality wassignificantly over 50% when toxin:peptide ratio reached 1:8 (57.3±1.0) %(P<0.05).

Full List of Delta-Endotoxins

Click on name to access NCBI entry (if available)

Name Acc No. Authors Year Source Strain Comment Cry1Aa1 M11250 Schnepfet al 1985 Bt kurstaki HD1 Cry1Aa2 M10917 Shibano et al 1985 Bt sottoCry1Aa3 D00348 Shimizu et al 1988 Bt aizawai IPL7 Cry1Aa4 X13535 Massonet al 1989 Bt entomocidus Cry1Aa5 D17518 Udayasuriyan et al 1994 BtFu-2-7 Cry1Aa6 U43605 Masson et al 1994 Bt kurstaki NRD-12 Cry1Aa7AF081790 Osman et al 1999 Bt C12 Cry1Aa8 I26149 Liu 1996 Cry1Aa9AB026261 Nagamatsu et al 1999 Bt dendrolimus T84A1 Cry1Aa10 AF154676 Houand Chen 1999 Bt kurstaki HD-1-02 Cry1Aa11 Y09663 Tounsi et al 1999 Btkurstaki Cry1Aa12 AF384211 Yao et al 2001 Bt Ly30 Cry1Aa13 AF510713Zhong et al 2002 Bt sotto Cry1Aa14 AY197341 Yingbo et al 2002unpublished Cry1Ab1 M13898 Wabiko et al 1986 Bt berliner 1715 Cry1Ab2M12661 Thorne et al 1986 Bt kurstaki Cry1Ab3 M15271 Geiser et al 1986 Btkurstaki HD1 Cry1Ab4 D00117 Kondo et al 1987 Bt kurstaki HD1 Cry1Ab5X04698 Hofte et al 1986 Bt berliner 1715 Cry1Ab6 M37263 Hefford et al1987 Bt kurstaki NRD-12 Cry1Ab7 X13233 Haider & Ellar 1988 Bt aizawaiIC1 Cry1Ab8 M16463 Oeda et al 1987 Bt aizawai IPL7 Cry1Ab9 X54939 Chak &Jen 1993 Bt aizawai HD133 Cry1Ab10 A29125 Fischhoff et al 1987 Btkurstaki HD1 Cry1Ab11 I12419 Ely & Tippett 1995 Bt A20 Cry1Ab12 AF059670Silva-Werneck et al 1998 Bt kurstaki S93 Cry1Ab13 AF254640 Tan et al2002 Bt c005 Cry1Ab14 U94191 Meza-Basso & 2000 Native Chilean BtTheoduloz Cry1Ab15 AF358861 Li, Zhang et al 2001 Bt B-Hm-16 Cry1Ab16AF375608 Yu et al 2002 Bt AC-11 Cry1Ab17 AAT46415 Huang et al 2004 BtWB9 Cry1Ab18 AAQ88259 Stobdan et al 2004 Bt Cry1Ab- AF327924Nagarathinam et al 2001 Bt kunthala RX24 uncertain sequence like Cry1Ab-AF327925 Nagarathinam et al 2001 Bt kunthala RX28 uncertain sequencelike Cry1Ab- AF327926 Nagarathinam et al 2001 Bt kunthala RX27 uncertainsequence like Cry1Ac1 M11068 Adang et al 1985 Bt kurstaki HD73 Cry1Ac2M35524 Von Tersch et al 1991 Bt kenyae Cry1Ac3 X54159 Dardenne et al1990 Bt BTS89A Cry1Ac4 M73249 Payne et al 1991 Bt kurstaki PS85A1Cry1Ac5 M73248 Payne et al 1992 Bt kurstaki PS81GG Cry1Ac6 U43606 Massonet al 1994 Bt kurstaki NRD-12 Cry1Ac7 U87793 Herrera et al 1994 Btkurstaki HD73 Cry1Ac8 U87397 Omolo et al 1997 Bt kurstaki HD73 Cry1Ac9U89872 Gleave et al 1992 Bt DSIR732 Cry1Ac10 AJ002514 Sun and Yu 1997 Btkurstaki YBT-1520 Cry1Ac11 AJ130970 Makhdoom & Riazuddin 1998 Cry1Ac12I12418 Ely & Tippett 1995 Bt A20 Cry1Ac13 AF148644 Qiao et al 1999 Btkurstaki HD1 Cry1Ac14 AF492767 Yao et al 2002 Bt Ly30 Cry1Ac15 AY122057Tzeng et al 2001 Bt from Taiwan Cry1Ad1 M73250 Payne & Sick 1993 Btaizawai PS811 Cry1Ad2 A27531 1995 Bt PS81RR1 Cry1Ae1 M65252 Lee &Aronson 1991 Bt alesti Cry1Af1 U82003 Kang et al 1997 Bt NT0423 Cry1Ag1AF081248 Mustafa 1999 Cry1Ah1 AF281866 Tan et al 2000 Cry1Ai1 AY174873Wang et al 2002 Cry1A-like AF327927 Nagarathinam et al 2001 Bt kunthalanags3 uncertain sequence Cry1Ba1 X06711 Brizzard & Whiteley 1988 Btthuringiensis HD2 Cry1Ba2 X95704 Soetaert 1996 Bt entomocidus HD110Cry1Ba3 AF368257 Zhang et al 2001 Cry1Ba4 AF363025 Mat Isa et al 2001 Btentomocidus HD9 Cry1Bb1 L32020 Donovan et al 1994 Bt EG5847 Cry1Bc1Z46442 Bishop et al 1994 Bt morrisoni Cry1Bd1 U70726 Kuo et al 2000 Btwuhanensis HD525 Cry1Bd2 AY138457 Isakova et al 2002 Bt 834 Cry1Be1AF077326 Payne et al 1998 Bt PS158C2 Cry1Be2 AAQ52387 Baum et al 2003Cry1Bf1 AX189649 Arnaut et al 2001 Cry1Bf2 AAQ52380 Baum et al 2003Cry1Bg1 AY176063 Wang et al 2002 Cry1Ca1 X07518 Honee et al 1988 Btentomocidus 60.5 Cry1Ca2 X13620 Sanchis et al 1989 Bt aizawai 7.29Cry1Ca3 M73251 Feitelson 1993 Bt aizawai PS81I Cry1Ca4 A27642 VanMellaert et al 1990 Bt entomocidus HD110 Cry1Ca5 X96682 Strizhov 1996 Btaizawai 7.29 Cry1Ca6 [1] AF215647 Yu et al 2000 Bt AF-2 Cry1Ca7 AY015492Aixing et al 2000 Cry1Ca8 AF362020 Chen et al 2001 Cry1Ca9 AY078160 Kaoet al 2003 Bt G10-01A Cry1Ca10 AF540014 Lin et al 2003 Bt Cry1Cb1 M97880Kalman et al 1993 Bt galleriae HD29 Cry1Cb2 AY007686 Song et al 2000Cry1Da1 X54160 Hofte et al 1990 Bt aizawai HD68 Cry1Da2 I76415 Payne &Sick 1997 Cry1Db1 Z22511 Lambert 1993 Bt BTS00349A Cry1Db2 AF358862 Liet al 2001 Bt B-Pr-88 Cry1Ea1 X53985 Visser et al 1990 Bt kenyae 4F1Cry1Ea2 X56144 Bosse et al 1990 Bt kenyae Cry1Ea3 M73252 Payne & Sick1991 Bt kenyae PS81F Cry1Ea4 U94323 Barboza-Corona et al 1998 Bt kenyaeLBIT-147 Cry1Ea5 A15535 Botterman et al 1994 Cry1Ea6 AF202531 Sun et al1999 Cry1Eb1 M73253 Payne & Sick 1993 Bt aizawai PS81A2 Cry1Fa1 M63897Chambers et al 1991 Bt aizawai EG6346 Cry1Fa2 M73254 Payne & Sick 1993Bt aizawai PS81I Cry1Fb1 Z22512 Lambert 1993 Bt BTS00349A Cry1Fb2AB012288 Masuda & Asano 1998 Bt morrisoni INA67 Cry1Fb3 AF062350 Song &Zhang 1998 Bt morrisoni Cry1Fb4 I73895 Payne et al 1997 Cry1Fb5 AF336114Li et al 2001 Bt B-Pr-88 Cry1Ga1 Z22510 Lambert 1993 Bt BTS0349A Cry1Ga2Y09326 Shevelev et al 1997 Bt wuhanensis Cry1Gb1 U70725 Kuo & Chak 1999Bt wuhanensis HD525 Cry1Gb2 AF288683 Li et al 2000 Bt B-Pr-88 Cry1GcAAQ52381 Baum et al 2003 Cry1Ha1 Z22513 Lambert 1993 Bt BTS02069AACry1Hb1 U35780 Koo et al 1995 Bt morrisoni BF190 Cry1H-like AF182196Srifah et al 1999 Bt JC291 insufficient sequence Cry1Ia1 X62821 Tailoret al 1992 Bt kurstaki Cry1Ia2 M98544 Gleave et al 1993 Bt kurstakiCry1Ia3 L36338 Shin et al 1995 Bt kurstaki HD1 Cry1Ia4 L49391 Kostichkaet al 1996 Bt AB88 Cry1Ia5 Y08920 Selvapandiyan 1996 Bt 61 Cry1Ia6AF076953 Zhong et al 1998 Bt kurstaki S101 Cry1Ia7 AF278797 Porcar 2000Bt Cry1Ia8 AF373207 Song et al 2001 Cry1Ia9 AF521013 Yao et al 2002 BtLy30 Cry1Ia10 AY262167 Espindola 2003 Bt thuringiensis Cry1Ia11 AJ315121Tounsi 2003 Bt kurstaki BNS3 Cry1Ib1 U07642 Shin et al 1995 Btentomocidus BP465 Cry1Ic1 AF056933 Osman et al 1998 Bt C18 Cry1Ic2AAE71691 Osman et al 2001 Cry1Id1 AF047579 Choi 2000 Cry1Ie1 AF211190Song et al 2000 Bt BTC007 Cry1If1 AAQ52382 Baum et al 2003 Cry1I-likeI90732 Payne et al 1998 insufficient sequence Cry1Ja1 L32019 Donovan etal 1994 Bt EG5847 Cry1Jb1 U31527 Von Tersch & Gonzalez 1994 Bt EG5092Cry1Jc1 I90730 Payne et al 1998 Cry1Jc2 AAQ52372 Baum et al 2003 Cry1Jd1AX189651 Arnaut et al 2001 Cry1Ka1 U28801 Koo et al 1995 Bt morrisoniBF190 Cry1La1 AAS60191 Je et al 2004 Bt kurstaki K1 Cry1-like I90729Payne et al 1998 insufficient sequence Cry2Aa1 M31738 Donovan et al 1989Bt kurstaki Cry2Aa2 M23723 Widner & Whiteley 1989 Bt kurstaki HD1Cry2Aa3 D86064 Sasaki et al 1997 Bt sotto Cry2Aa4 AF047038 Misra et al1998 Bt kenyae HD549 Cry2Aa5 AJ132464 Yu & Pang 1999 Bt SL39 Cry2Aa6AJ132465 Yu & Pang 1999 Bt YZ71 Cry2Aa7 AJ132463 Yu & Pang 1999 Bt CY29Cry2Aa8 AF252262 Wei et al 2000 Bt Dongbei 66 Cry2Aa9 AF273218 Zhang etal 2000 Cry2Aa10 AF433645 Yao et al 2001 Cry2Aa11 AAQ52384 Baum et al2003 Cry2Ab1 M23724 Widner & Whiteley 1989 Bt kurstaki HD1 Cry2Ab2X55416 Dankocsik et al 1990 Bt kurstaki HD1 Cry2Ab3 AF164666 Chen et al1999 Bt BTC002 Cry2Ab4 AF336115 Li et al 2001 Bt B-Pr-88 Cry2Ab5AF441855 Yao et al 2001 Cry2Ab6 AY297091 Wang et al 2003 Bt WZ-7 Cry2Ac1X57252 Wu et al 1991 Bt shanghai S1 Cry2Ac2 AY007687 Song et al 2000Cry2Ac3 AAQ52385 Baum et al 2003 Cry2Ad1 AF200816 Choi et al 1999 BtBR30 Cry2Ae1 AAQ52362 Baum et al 2003 Cry3Aa1 M22472 Herrnstadt et al1987 Bt san diego Cry3Aa2 J02978 Sekar et al 1987 Bt tenebrionis Cry3Aa3Y00420 Hofte et al 1987 Cry3Aa4 M30503 McPherson et al 1988 Bttenebrionis Cry3Aa5 M37207 Donovan et al 1988 Bt morrisoni EG2158Cry3Aa6 U10985 Adams et al 1994 Bt tenebrionis Cry3Aa7 AJ237900 Zhang etal 1999 Bt 22 Cry3Aa8 AAS79487 Gao and Cai 2004 Bt YM-03 Cry3Aa9AAW05659 Bulla and Candas 2004 Bt UTD-001 Cry3Aa10 AAU29411 Chen et al2004 Bt 886 Cry3Ba1 X17123 Sick et al 1990 Bt tolworthi 43F Cry3Ba2A07234 Peferoen et al 1990 Bt PGSI208 Cry3Bb1 M89794 Donovan et al 1992Bt EG4961 Cry3Bb2 U31633 Donovan et al 1995 Bt EG5144 Cry3Bb3 I15475Peferoen et al 1995 Cry3Ca1 X59797 Lambert et al 1992 Bt kurstakiBtI109P Cry4Aa1 Y00423 Ward & Ellar 1987 Bt israelensis Cry4Aa2 D00248Sen et al 1988 Bt israelensis HD522 Cry4Aa3 AL731825 Berry et al 2002 Btisraelensis Cry4Ba1 X07423 Chungjatpornchai et al 1988 Bt israelensis4Q2-72 Cry4Ba2 X07082 Tungpradubkul et al 1988 Bt israelensis Cry4Ba3M20242 Yamamoto et al 1988 Bt israelensis Cry4Ba4 D00247 Sen et al 1988Bt israelensis HD522 Cry4Ba5 AL731825 Berry et al 2002 Bt israelensisCry5Aa1 L07025 Narva et al 1994 Bt darmstadiensis PS17 Cry5Ab1 L07026Narva et al 1991 Bt darmstadiensis PS17 Cry5Ac1 I34543 Payne et al 1997Cry5Ba1 U19725 Foncerrada &Narva 1997 Bt PS86Q3 Cry6Aa1 L07022 Narva etal 1993 Bt PS52A1 Cry6Aa2 AF499736 Bai et al 2001 Bt YBT1518 Cry6Ba1L07024 Narva et al 1991 Bt PS69D1 Cry7Aa1 M64478 Lambert et al 1992 Btgalleriae PGSI245 Cry7Ab1 U04367 Payne & Fu 1994 Bt dakota HD5I1 Cry7Ab2U04368 Payne & Fu 1994 Bt kumamotoensis 867 Cry8Aa1 U04364 Narva & Fu1992 Bt kumamotoensis Cry8Ba1 U04365 Narva & Fu 1993 Bt kumamotoensisCry8Bb1 AX543924 Abad et al 2002 Cry8Bc1 AX543926 Abad et al 2002Cry8Ca1 U04366 Ogiwara et al. 1995 Bt japonensis Buibui Cry8Ca2 AAR98783Song et al 2004 Bt HBF-1 Cry8Da1 AB089299 Yamamoto & Asano 2002 Btgalleriae Cry8Da2 BD133574 Asano et al 2002 Bt Cry8Da3 BD133575 Asano etal 2002 Bt Cry8Ea1 AY329081 Fuping et al 2003 Bt 185 Cry8Fa1 AY551093Fuping et al 2004 Bt 185 No NCBI link yet Cry1Ga1 AY590188 Fuping et al2004 Bt HBF-18 No NCBI link yet Cry9Aa1 X58120 Smulevitch et al 1991 Btgalleriae Cry9Aa2 X58534 Gleave et al 1992 Bt DSIR517 Cry9Aa likeAAQ52376 Baum et al 2003 incomplete sequence Cry9Ba1 X75019 Shevelev etal 1993 Bt galleriae Cry9Bb1 AY758316 Silva-Werneck et al 2004 Btjaponensis Cry9Ca1 Z37527 Lambert et al 1996 Bt tolworthi Cry9Ca2AAQ52375 Baum et al 2003 Cry9Da1 D85560 Asano et al 1997 Bt japonensisN141 Cry9Da2 AF042733 Wasano & Ohba 1998 Bt japonensis Cry9Ea1 AB011496Midoh & Oyama 1998 Bt aizawai SSK-10 Cry9Ea2 AF358863 Li et al 2001 BtB-Hm-16 Cry9Eb1 AX189653 Arnaut et al 2001 Cry9Ec1 AF093107 Wasano &Ohba 2003 Bt galleriae Cry9 like AF093107 Wasano et al 1998 Bt galleriaeinsufficient sequence Cry10Aa1 M12662 Thorne et al 1986 Bt israelensisCry10Aa2 E00614 Aran & Toomasu 1996 Bt israelensis ONR- 60A Cry10Aa3AL731825 Berry et al 2002 Bt israelensis Cry11Aa1 M31737 Donovan et al1988 Bt israelensis Cry11Aa2 M22860 Adams et al 1989 Bt israelensisCry11Aa3 AL731825 Berry et al 2002 Bt israelensis Cry11Ba1 X86902Delecluse et al 1995 Bt jegathesan 367 Cry11Bb1 AF017416 Orduz et al1998 Bt medellin Cry12Aa1 L07027 Narva et al 1991 Bt PS33F2 Cry13Aa1L07023 Narva et al 1992 Bt PS63B Cry14Aa1 U13955 Narva et al 1994 Btsotto PS80JJ1 Cry15Aa1 M76442 Brown & Whiteley 1992 Bt thompsoniCry16Aa1 X94146 Barloy et al 1996 Cb malaysia CH18 Cry17Aa1 X99478Barloy et al 1998 Cb malaysia CH18 Cry18Aa1 X99049 Zhang et al 1997Paenibacillus popilliae Cry18Ba1 AF169250 Patel et al 1999 Paenibacilluspopilliae Cry18Ca1 AF169251 Patel et al 1999 Paenibacillus popilliaeCry19Aa1 Y07603 Rosso & Delecluse 1996 Bt jegathesan 367 Cry19Ba1 D88381Hwang et al 1998 Bt higo Cry20Aa1 U82518 Lee & Gill 1997 Bt fukuokaensisCry21Aa1 I32932 Payne et al 1996 Cry21Aa2 I66477 Feitelson 1997 Cry21Ba1AB088406 Sato & Asano 2002 Bt roskildiensis Cry22Aa1 I34547 Payne et al1997 Cry22Aa2 AX472772 Isaac et al 2002 Bt Cry22Ab1 AAK50456 Baum et al2000 Bt EG4140 Cry22Ab2 AX472764 Isaac et al 2002 Bt Cry22Ba1 AX472770Isaac et al 2002 Bt Cry23Aa1 AAF76375 Donovan et al 2000 Bt Binary withCry37Aa1 Cry24Aa1 U88188 Kawalek and Gill 1998 Bt jegathesan Cry24Ba1BAD32657 Ohgushi et al 2004 Bt sotto Cry25Aa1 U88189 Kawalek and Gill1998 Bt jegathesan Cry26Aa1 AF122897 Wojciechowska et al 1999 Btfinitimus B-1166 Cry27Aa1 AB023293 Saitoh 1999 Bt higo Cry28Aa1 AF132928Wojciechowska et al 1999 Bt finitimus B-1161 Cry28Aa2 AF285775 Moore andDebro 2000 Bt finitimus Cry29Aa1 AJ251977 Delecluse et al 2000 Cry30Aa1AJ251978 Delecluse et al 2000 Cry30Ba1 BAD00052 Ikeya et al 2003 Btentomocidus Cry30Ca1 BAD67157 Ohgushi et al 2004 Bt sotto Cry31Aa1AB031065 Mizuki et al 2000 Bt 84-HS-1-11 Cry31Aa2 AY081052 Jung and Cote2000 Bt Cry32Aa1 AY008143 Balasubramanian et al 2001 Bt yunnanensisCry32Ba1 BAB78601 Takebe et al 2001 Bt Cry32Ca1 BAB78602 Takebe et al2001 Bt Cry32Da1 BAB78603 Takebe et al 2001 Bt Cry33Aa1 AAL26871 Kim etal 2001 Bt dakota Cry34Aa1 AAG50341 Ellis et al 2001 Bt PS80JJ1 Binarywith Cry35Aa1 Cry34Aa2 AAK64560 Rupar et al 2001 Bt EG5899 Binary withCry35Aa2 Cry34Aa3 AY536899 Schnepf et al 2004 Cry34Aa4 AY536897 Schnepfet al 2004 Cry34Ab1 AAG41671 Moellenbeck et al 2001 Bt PS149B1 Binarywith Cry35Ab1 Cry34Ac1 AAG50118 Ellis et al 2001 Bt PS167H2 Binary withCry35Ac1 Cry34Ac2 AAK64562 Rupar et al 2001 Bt EG9444 Binary withCry35Ab2 Cry34Ac3 AY536896 Schnepf et al 2004 Cry34Ba1 AAK64565 Rupar etal 2001 Bt EG4851 Binary with Cry35Ba1 Cry34Ba2 AY536900 Schnepf et al2004 Cry34Ba3 AY536898 Schnepf et al 2004 Cry35Aa1 AAG50342 Ellis et al2001 Bt PS80JJ1 Binary with Cry34Aa1 Cry35Aa2 AAK64561 Rupar et al 2001Bt EG5899 Binary with Cry34Aa2 Cry35Aa3 AY536895 Schnepf et al 2004Cry35Aa4 AY536892 Schnepf et al 2004 Cry35Ab1 AAG41672 Moellenbeck et al2001 Bt PS149B1 Binary with Cry34Ab1 Cry35Ab2 AAK64563 Rupar et al 2001Bt EG9444 Binary with Cry34Ac2 Cry35Ab3 AY536891 Schnepf et al 2004Cry35Ac1 AAG50117 Ellis et al 2001 Bt PS167H2 Binary with Cry34Ac1Cry35Ba1 AAK64566 Rupar et al 2001 Bt EG4851 Binary with Cry34Ba1Cry35Ba2 AY536894 Schnepf et al 2004 Cry35Ba3 AY536893 Schnepf et al2004 Cry36Aa1 AAK64558 Rupar et al 2001 Bt Cry37Aa1 AAF76376 Donovan etal 2000 Bt Binary with Cry23Aa Cry38Aa1 AAK64559 Rupar et al 2000 BtCry39Aa1 BAB72016 Ito et al 2001 Bt aizawai Cry40Aa1 BAB72018 Ito et al2001 Bt aizawai Cry40Ba1 BAC77648 Ito et al 2003 Bun1-14 Cry41Aa1AB116649 Yamashita et al 2003 Bt A1462 Cry41Ab1 AB116651 Yamashita et al2003 Bt A1462 Cry42Aa1 AB116652 Yamashita et al 2003 Bt A1462 Cry43Aa1AB115422 Yokoyama and Tanaka 2003 P. lentimorbus semadara Cry43Aa2AB176668 Nozawa 2004 P. popilliae popilliae No NCBI link yet Cry43Ba1AB115422 Yokoyama and Tanaka 2003 P. lentimorbus semadara Cry43-likeAB115422 Yokoyama and Tanaka 2003 P. lentimorbus semadara Cry44AaBAD08532 Ikeya et al 2004 Bt entomocidus INA288 Cry45Aa BAD22577 Okumuraand Saitoh 2004 Bt 89-T-34-22 Cry46Aa BAC79010 Ito et al 2004 Bt dakotaCry46Ab BAD35170 Yamagiwa et al 2004 Bt Cyt1Aa1 X03182 Waalwijk et al1985 Bt israelensis Cyt1Aa2 X04338 Ward & Ellar 1986 Bt israelensisCyt1Aa3 Y00135 Earp & Ellar 1987 Bt morrisoni PG14 Cyt1Aa4 M35968Galjart et al 1987 Bt morrisoni PG14 Cyt1Aa5 AL731825 Berry et al 2002Bt israelensis Cyt1Ab1 X98793 Thiery et al 1997 Bt medellin Cyt1Ba1U37196 Payne et al 1995 Bt neoleoensis Cyt1Ca1 AL731825 Berry et al 2002Bt israelensis unusual hybrid Cyt2Aa1 Z14147 Koni & Ellar 1993 Btkyushuensis Cyt2Aa2 AF472606 Promdonkoy & Panyim 2001 Bt darmstadiensis73E10 Cyt2Ba1 U52043 Guerchicoff et al 1997 Bt israelensis 4Q2 Cyt2Ba2AF020789 Guerchicoff et al 1997 Bt israelensis PG14 Cyt2Ba3 AF022884Guerchicoff et al 1997 Bt fuokukaensis Cyt2Ba4 AF022885 Guerchicoff etal 1997 Bt morrisoni HD12 Cyt2Ba5 AF022886 Guerchicoff et al 1997 Btmorrisoni HD518 Cyt2Ba6 AF034926 Guerchicoff et al 1997 Bt tenebrionisCyt2Ba7 AF215645 Yu & Pang 2000 Bt T301 Cyt2Ba8 AF215646 Yu & Pang 2000Bt T36 Cyt2Ba9 AL731825 Berry et al 2002 Bt israelensis Cyt2Bb1 U82519Cheong & Gill 1997 Bt jegathesan Cyt2Bc1 CAC80987 Delecluse et al 1999Bt medellin Cyt2Ca1 AAK50455 Baum et al 2001 Bt Footnotes [1] Thesequences for toxins orginally designated Cry1Ca6 and Cry1Ca7 (Crickmoreet al 1998 Microbiol. Mol. Biol. Rev.62: 807-813) were subsequentlywithdrawn by the database managers.

Some of the above toxins could not be given an unambiguous name due toinsufficient or uncertain sequence data.

The following Bt proteins have not been assigned a name or entered intothe nomenclature for the reasons given.

Name Accession Reference Year Source Strain Reason 40 kDa AAA22332 Brownand 1992 Bt thompsoni No reported toxicity Whiteley NT32KD AAL26870 Kimet al 2001 Bt dakota No reported toxicity CryC35 CAA63374 Juarez-Perezet al 1995 Bt cameroun No reported toxicity 273B CryTDK BAA13073Hashimoto 1996 Bt mexicanensis S-layer protein, not toxin? CryC53CAA67205 Juarez-Perez et al 1996 Bt cameroun No reported toxicity 273BMTx2 p21med CAA67329 Thiery et al 1997 Bt 163-131 chaperone, not toxin?

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1. A method of inhibiting an insect wherein said method comprisesproviding a peptide to said insect for ingestion, wherein said peptideenhances toxin activity of a Bacillus thuringiensis Cry protein, whereinsaid peptide comprises a B.t. Cry binding domain of an ectodomain of aninsect cadherin B.t. toxin receptor from an insect midgut epithelialcell, and wherein said peptide is a fragment of said ectodomain.
 2. Themethod of claim 1, wherein said method further comprises providing aBacillus thuringiensis Cry protein to said insect for ingestion.
 3. Amethod of inhibiting an insect wherein said method comprises providing apeptide to said insect for ingestion, wherein said peptide binds aBacillus thuringiensis Cry protein and enhances toxin activity of saidprotein, and wherein said peptide comprises a first B.t. Cry bindingdomain having at least 85% identity with a second B.t. Cry bindingdomain of an ectodomain of an insect cadherin B.t. toxin receptor froman insect midgut epithelial cell.
 4. The method of claim 3, wherein saidpeptide is sprayed on to a plant.
 5. The method of claim 3, wherein saidpeptide is produced by and present in a plant.
 6. The method of claim 3,wherein said method further comprises providing said Bacillusthuringiensis Cry protein to said insect for ingestion.
 7. The method ofclaim 6, wherein said peptide is sprayed on to a plant that producessaid protein.
 8. The method of claim 6, wherein said peptide and saidprotein are sprayed on to a plant.
 9. The method of claim 6, whereinsaid peptide and said protein are produced by and are present in aplant.
 10. The method of claim 6, wherein said peptide is fused withsaid Cry protein.
 11. The method of claim 1, wherein said peptide issprayed on to a plant.
 12. The method of claim 1, wherein said peptideis produced by and present in a plant.
 13. The method of claim 1,wherein said method further comprises providing said Bacillusthuringiensis Cry protein to said insect for ingestion.
 14. The methodof claim 13, wherein said peptide is sprayed on to a plant that producessaid protein.
 15. The method of claim 13, wherein said peptide and saidprotein are sprayed on to a plant.
 16. The method of claim 13, whereinsaid peptide and said protein are produced by and are present in aplant.
 17. The method of claim 3, wherein said first B.t. Cry bindingdomain has at least 90% identity with said second B.t. Cry bindingdomain.
 18. The method of claim 3, wherein said first B.t. Cry bindingdomain has at least 95% identity with said second B.t. Cry bindingdomain.
 19. The method of claim 1, wherein said peptide is fused with aprotein.
 20. A fusion protein comprising a peptide fused to a fusedprotein, wherein said peptide binds a Bacillus thuringiensis Cry proteinand enhances toxin activity of said Bacillus thuringiensis Cry protein,and wherein said peptide comprises a B.t. Cry binding domain having atleast 85% identity with a B.t. Cry binding domain of an ectodomain of aninsect cadherin B.t. toxin receptor from an insect midgut epithelialcell.
 21. The fusion protein of claim 20, wherein said fused protein issaid Bacillus thuringiensis Cry protein.